WO2013087921A1

WO2013087921A1 - Engineered fluorescent proteins for enhanced fret and uses thereof

Info

Publication number: WO2013087921A1
Application number: PCT/EP2012/075737
Authority: WO
Inventors: Raik GRÜNBERG; Luis Serrano Pubul; François STRICHER
Original assignee: Fundació Privada Centre De Regulació Genòmica (Crg); Institució Catalana De Recerca I Estudis Avançats (Icrea)
Priority date: 2011-12-15
Filing date: 2012-12-17
Publication date: 2013-06-20
Also published as: GB201121565D0

Abstract

The invention relates to pairs of genetically engineered fluorescent proteins and their uses in FRET- based applications, as well as methods to produce genetically engineered fluorescent proteins for high efficiency FRET (Forster Resonance Energy Transfer) based on the introduction of electrostatic helper interactions.

Description

ENGINEERED FLUORESCENT PROTEINS FOR ENHANCED FRET AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to the field of fluorescent proteins and their versatile applications in the field of molecular biology. More specifically, the invention relates to pairs of genetically engineered fluorescent proteins and their uses in FRET-based applications, as well as methods to produce genetically engineered fluorescent proteins for high efficiency FRET (Forster Resonance Energy Transfer) based on the introduction of electrostatic helper interactions.

BACKGROUND

Fluorescent proteins are widely known today for their use as fluorescent markers in biomedical sciences. They are applied for a wide range of applications including the study of gene expression, protein localization, visualizing subcellular organelles in cells, visualizing protein localization and transport, as well as for detecting protein-protein interactions, or for screening purposes, amongst many others. Due to the potential for widespread usage and the evolving needs of researchers, novel fluorescent proteins have been identified with improved fluorescence intensity and maturation rates at physiological temperatures, modified excitation and emission spectra, and reduced oligomerization and aggregation properties. In addition, mutagenesis of known proteins has been undertaken to improve their chemical properties. Finally, codon usage has been optimized for high expression in the desired heterological system, for example in mammalian cells.

Among the different methods for the detection of protein-protein interactions, FRET (Forster Resonance Energy Transfer) between genetically encoded fluorescent proteins [1] has several distinct advantages: molecular interactions can be detected in real time, both in vitro or in live cells, and without the need of co-factors or auxiliary reactions. FRET is fully reversible and reports both on the formation or the disruption of interactions - an important difference to other popular assays, for example, based on protein fragment complementation [2]. This, as well as the possibility to visualize interactions at sub-cellular spatial resolution, makes FRET ideally suited for the study of signaling and many other dynamic processes within cells, animals or in vitro systems.

Hypothetically, the interaction between any two proteins of interest could be monitored with the aid of two simple protein fusions: one protein is fused to a donor and the other to the acceptor fluorophore. Protein-based FRET probes are relatively bulky. Long and flexible peptide linkers between the fluorophore and the protein of interest are thus preferable in order to minimize any interference of the probe with the interaction of interest. However, energy transfer from donor to acceptor fluorophores only occurs over very short distances. As illustrated in Figure la, already a moderate separation by 10 to 12 nm - about twice the length of the fluorescent protein barrel - diminishes the signal to about 1% FRET efficiency. FRET probe engineering is therefore caught between the two opposing requirements of sufficiently long linkers versus short reach. A compromise needs to be found case by case and this has, until now, severely limited the general application of the approach.

Enhanced protein-based FRET pairs have so far been mostly a product of the ongoing efforts to improve spectral properties of individual fluorescent proteins (as outlined hereinbefore). Photostability, higher quantum yields, single absorption peaks and better separation between donor and acceptor emission spectra all facilitate the measurement of FRET[3]. Nevertheless, these separate improvements cannot address the limitation of FRET by the strong dependence on fluorophore distances and orientations. Few studies tried to optimize a pair of donor and acceptor proteins simultaneously. The CyPet/YPet pair was developed by directed evolution and FACS screening from the cyan and yellow fluorescent proteins ECFP and EYFP[4]. The two proteins were connected through a flexible protease cleavage site and optimized for high FRET change between uncleaved and cleaved state. ECFP and EYFP are both derived from Aequorea GFP with a tendency to homodimerize (K_D ~ 0.1 M) [5]. The large improvement in FRET was later shown to be caused mainly by only two mutations that re-enforce the native homodimerization interface stemming from Aequorea GFP [6, 7] - leading to the formation of a high FRET intramolecular complex between CyPet and YPet. Such direct interactions among fluorescent proteins are traditionally considered annoyance [5] rather than virtue. They are, however, the best way to multiply FRET responses, in particular, if sensors are based on differences between high (bound, connected) and low (unbound, cleaved) local concentrations of donor and acceptor [8]. Also unimolecular FRET constructs can benefit from the weak dimerization of GFP-derived donor and acceptor domains [7, 9].

The interaction between GFP-derived donor and acceptor domains can be rationally enhanced or weakened through the introduction of point mutations among the residues that mediate the intermolecular contact [6,7 and published patent application WO 97/28261]. These residues can be inferred from the crystal structure of the GFP homodimer. Enhanced dimerization can be achieved through the introduction of additional hydrophobic contacts or the introduction of complementary electrostatic charges or both. Electrostatic charges can also be added indirectly, by engineering metal ion binding sites within the dimerization interface [40]. However, all these approaches have several disadvantages. First, as they rely on improving the native GFP homodimerization interface, these methods are limited to pairs of fluorescent proteins where both donor and acceptor are derived from Aequorea GFP. Such cyan / yellow or related FRET pairs are not without problems: the multi- exponential decay kinetics of most cyan proteins [3] complicates fluorescence lifetime measurements and the short excitation wavelength of the cyan donor provokes background from cellular autofluorescence. Secondly, the similarity of GFP-derived donor and acceptor proteins leads to a nearly symmetric interface. Owing to this symmetry, any increase in donor/acceptor heterodimerization is typically accompanied by increased donor/donor and acceptor/acceptor homodimerization. Vice versa, mutations that counter donor/donor or acceptor/acceptor homodimerization [5] also abolish the donor/acceptor heterodimerization [9]. Potentially beneficial heterodimerization thus has to be balanced against unwanted homodimerization tendencies. Thirdly, the introduction of molecular contacts within the interface stabilizes the formation of an actual donor/acceptor complex with reduced off rate or, correspondingly, a longer life time and tighter binding. Increased affinities can lead to undesired background signal of a FRET sensor which may eliminate any advantage of overall FRET increase [9]. Increased binding and life times may also affect the switching dynamics and reversibility of FRET sensors.

Thus, there is still a need for alternative FRET pairs with increased FRET efficiency and less dependency on fluorophore distances and orientations. Ideally, such pairs: (1) would be excited at longer wavelengths in order to reduce phototoxicity and autofluorescence and increase the penetrance of emission light; (2) would be composed of two un-related fluorescent proteins so that heterodimerization would not translate to homodimerization of acceptor or donor; (3) would work by creating a weakly aligned donor/acceptor encounter complex without actual binding, that means, they should show high on rates but very low half live of the donor/acceptor arrangement; SUMMARY OF THE INVENTION

In order to overcome the current problems with existing FRET probes, different novel strategies were developed for the rational design of weak but strictly heterodimeric interactions ("helper interactions") between fluorescent proteins. We designate these methods "helper interaction FRET" (hiFRET). One of these methods is based on computational protein design and allowed us to create interfaces for electrostatically driven encounter complexes between unrelated fluorescent proteins. Using this strategy, we developed a set of red-shifted FRET pairs optimized for the robust detection of protein- protein interactions. Our helper interaction(s) led to a large increase of FRET efficiency while background signals in the unbound state remained near zero. The enhanced red-shifted FRET pairs will be of immediate use for in vitro and in vivo FRET-based applications. They provide an attractive alternative to classic CFP/YFP pairs. Lack of background binding combined with low or absent homodimerization and red-shifted excitation and emission makes this pair well suited for in vitro assays where weak FRET signals may need to be amplified from complex mixtures across a wide range of protein concentrations. Moreover, since mCitrine / mCherry prove to be an optimal choice for excitation lifetime measurements due to monoexponential donor decay and very limited sensitized emission, such spectroscopic properties combined with helper interaction-induced signal enhancements should be of particular interest for upcoming large-scale FLIM experiments [22]. Accordingly, FRET signals between more than 14 synthetic protein variants were characterized in vitro by donor- and acceptor-based fluorescence intensity and were validated by lifetime (FLIM) measurements.

Thus, according to one aspect, the invention relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a. Providing a pair of fluorescent proteins suitable as a donor and acceptor for FRET measurements,

b. Introducing complementary electrostatic charges in said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins,

c. Measuring and comparing the FRET efficiency of said pair of engineered fluorescent proteins relative to the non-engineered pair in a suitable assay,

d. Selecting a pair of engineered fluorescent proteins having increased FRET efficiency relative to the non-engineered pair.

According to a specific embodiment, step b in the above described method is further characterized in that it comprises the steps of: a. Creating an in silico structural model of a heterodimeric complex of said pair of fluorescent proteins,

b. Introducing and selecting mutations that introduce complementary electrostatic charges within or, preferably, in the vicinity of the contact interface of said model heterodimeric complex of said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins.

The methods according to the invention as described hereinbefore may further comprise the steps of introducing mutations that remove unfavorable contacts within the contact interface of said model heterodimeric complex, and/or introducing mutations that disrupt homodimer interfaces without impairing heterodimerisation. The step of introducing complementary electrostatic charges in any of the above described methods may be further defined as introducing at least one amino acid substitution in at least one of the fluorescent proteins of the pair, preferably at least one positive charge in one fluorescent protein and at least one negative charge in the other fluorescent protein.

Preferably, the fluorescent proteins of the pair suitable as a donor and acceptor for FRET measurements to be provided in any of the above described methods do not show initial intrinsic interaction. In particular, said pair of fluorescent proteins may comprise (m)Citrine (SEQ ID NO: 1), or a variant thereof, and mCherry (SEQ ID NO: 4), or a variant thereof. More preferably, said pair of engineered fluorescent proteins may comprise: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1, selected from the group consisting of positions 43, 144, 202, 221 and 227, and

b. an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the group consisting of positions 39, 144, 145 and 196. wherein the at least one amino acid substitution may be selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: 1, and from the group consisting of E39K, E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.

Another aspect of the present invention relates to a pair of engineered fluorescent proteins having an increased FRET efficiency relative to a pair of SEQ ID NO: 1 and SEQ ID NO: 4, comprising a donor and an acceptor fluorescent protein, wherein said donor and acceptor fluorescent proteins may be chosen from the group consisting of: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein corresponding to SEQ ID NO: 4;

b. a donor fluorescent protein corresponding to SEQ ID NO: 1, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196.

c. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196;

d. variants of said donor and acceptor fluorescent proteins of (a), (b), or (c).

According to specific embodiments, the at least one amino acid substitution in the above described pair of engineered fluorescent proteins may be selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: 1, and from the group consisting of E39 , E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.

Further, a pair of engineered fluorescent proteins wherein the donor and the acceptor fluorescent proteins of the present invention are each fused to a polypeptide of interest, optionally through a linker molecule, is also provided. Accordingly, bimolecular and unimolecular constructs are also encompassed in the present invention.

In a preferred embodiment, the invention provides a bimolecular construct, comprising: a. a donor fluorescent protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules, and

b. an acceptor fluorescent protein of the invention fused to a polypeptide of interest, optionally through one or more linker molecules.

According to another preferred embodiment, the invention provides a unimolecular protein construct selected from the group comprising the following fusion proteins: a. a donor fluorescent protein - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, or

b. an acceptor fluorescent protein - a polypeptide of interest - a donor fluorescent protein, optionally fused through one or more linker molecules, or

c. a polypeptide of interest - a donor fluorescent protein - an acceptor fluorescent protein - a polypeptide of interest, optionally fused through one or more linker molecules, or

d. a polypeptide of interest - an acceptor fluorescent protein - a donor fluorescent protein - a polypeptide of interest, optionally fused through one or more linker molecules, or

e. a donor fluorescent protein - a polypeptide of interest - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, f. an acceptor fluorescent protein - a polypeptide of interest - a polypeptide of interest - a donor fluorescent protein, optionally fused through one or more linker molecules, and, wherein said donor and acceptor fluorescent proteins are according to the invention. Alternative embodiments of the invention encompass (1) an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, such as T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D; (2) an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144 and 145, such as E39 , E144K, E144R, A145K, A145R, N196K and N196R; (3) a fusion protein comprising an engineered fluorescent protein as defined in (1) or (2) fused to a polypeptide of interest, optionally through one or more linker molecules.

Further, polynucleotides encoding any of the donor and/or acceptor fluorescent proteins, or any of the engineered fluorescent proteins, or any of the bimolecular/unimolecular constructs, or any of the fusion proteins, according to the present invention, are also envisaged here, as well as expression vectors comprising suitable expression control sequences operably linked to any of the above described polynucleotides, as well as host cells comprising any of the polynucleotides or any of the expression vectors of the invention, as well as antibodies that specifically bind to the engineered fluorescent proteins. In a further aspect, the invention provides a kit comprising any of the polynucleotides or any of the expression vectors of the invention as described hereinbefore.

Still another aspect of the invention relates to the use of the pair of engineered fluorescent proteins, or the protein constructs, or the individual engineered fluorescent proteins, or the fusion proteins, or any of the polynucleotides or any of the expression vectors, all as defined hereinbefore, for in vitro and/or in vivo FRET-based applications.

Preferably, a method of identifying a specific interaction of a first polypeptide of interest and a second polypeptide of interest is provided, the method comprising: a. Contacting the first polypeptide of interest, which is fused to a donor fluorescent protein of a pair of engineered fluorescent proteins according to the invention, and the second polypeptide of interest, which is fused to the corresponding acceptor fluorescent protein of the pair of engineered fluorescent proteins according to the invention, under conditions that allow a specific interaction of the first and the second polypeptide of interest,

b. Exciting the donor, and

c. Detecting FRET from the donor to the acceptor, thereby identifying a specific interaction of the first and the second polypeptide of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l:Principle of enhanced FRET.(a) FRET signals are highly dependent on the distance between donor and acceptor. The efficiency of FRET at various distances is compared using two Citrine structures (drawn to scale) spaced at a distance at which 90% of the signal is lost (E=10%). The Forster distance was set to 56.6 A [12]. (b, c) Schematic description of protein interaction detection by FRET between conventional (b) and enhanced (c) FRET probes. The drug-induced interaction between FRB and FKBP12 (gray) is used to quantify FRET signals in unbound and bound state.

Figure 2:Design of FRET helper interactions. (a)Design of electrostatic encounter complexes. Amenable interface rim positions (green), but not interface core positions (gray), were screened in silico for mutations into complementary charged side chains. The (imperfect) template interface is shown from the side (center) and opened by 90 (left and right). The electrostatic potential (+0.5 and -0.5 k_b/T e) calculated at 150 mM ionic strength is shown below (right and left) with Cherry and Citrine in the same open orientation or, (middle) color-coded on the molecular surface of the complex, (b) Final design with positively charged side chains (blue) added to Cherry and negative charges (red) added to Citrine.

Figure 3:Enhanced FRET. Electrostatically enhanced FRET probes (shades of blue) yield higher FRET efficiencies than conventional probes (gray) and are less affected by prolongation of the linker with the FRB/FKBP12 reference interaction.

Figure 4. Emission spectra (top) and fluorescence lifetime (bottom) of conventional and enhanced

FRET pairs. Top: The best performing FRET pairs were mixed at 1:1 stoichiometry (0.5 μΜ, 24aa linker between binding domain and FP) and their interaction induced with rapamycin. The performance of the conventional FRET pair is shown in gray (unmodified Citrine / mCherry). Quenching of the donor emission by FRET is apparent for all pairs and is more pronounced for constructs with FRET helper interactions. Note, owing to errors in protein concentrations, exact 1:1 ratios are difficult to attain.

Quantitative FRET efficiencies were therefore determined in experiments with full donor occupancy.

All spectra are averages of three to six replicates and are normalized to the peak fluorescence (530 nm) of the donor-only samples. Bottom: Citrine fluorescence decay traces from the equivalent experiments on a FLIM microscope. Donor and acceptor were mixed at 0.3 and 0.5 μΜ concentrations, respectively, to ensure full donor occupancy. Raw data are shown in gray and overlaid with fits to a simple two- exponential model.

Figure 5.Specificity and background activity of helper interactions.(a) The FRET probe pairs with 24 aa linker were tested at high protein concentrations (3 μΜ [donor] + 5 μΜ [acceptor]). Gains of FRET efficiency are preserved, (b) FRET efficiencies with mismatched helper interactions (0.3 / 0.5 μΜ standard concentration). Partial FRET gains were achieved by combining engineered mCherry with non- engineered mCitrine.

Figure 6. Correlation of in-vitro FRET efficiencies measured from changes in donor or acceptor intensities. The slope of the acceptor: donor correlation is directly depending on the ratio of acceptor and donor extinction coefficients, which can only be measured with limited accuracy (and were assumed to be the same for all proteins). The broken line indicates linear least-square fits with the regression parameters given below.

Figure 7.ln-vitro FRET efficiencies measured at varying salt concentrations. Electrostatic helper interactions were diminished by increases in salt concentrations above physiological levels. Error bars indicate standard deviations from 5 or 6replicates.

Figure 8. Comparison of the original ("wild type") and engineered Citrine sequences. Blue arrows mark positions subject to mutations for the electrostatic interface. Unstructured peptide sequences introduced during the engineering of the original Citrine are labelled as 'spacer'. Additional (wt and 4-) variants with the homodimer-breaking mutation A206K (shown in red without arrow) are omitted for clarity.

Figure 9. Comparison of the original mCherry sequence with the two variants engineered for electrostatically enhanced FRET. Blue arrows mark positions subject to mutations. Unstructured peptide sequences introduced during the engineering of the original mCherry are labelled as 'spacer'. DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of molecular and cellular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Leach, Molecular Modelling: Principles and Applications, 2d ed., Prentice Hall, New Jersey(2001). Definitions

As used herein, the terms "determining," "measuring," "assessing," "testing", and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the terms "polypeptide", "protein", "peptide", "oligopeptide" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger NA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated NA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid vector", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include, without the purpose of being limitative, cosmids and yeast artificial chromosomes (YAC). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of certain genes of interest. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired.

The term "operably linked" as used herein refers to a linkage in which the regulatory sequence is contiguous with the gene of interest to control said gene of interest, as well as regulatory sequences that act in trans or at a distance to control the gene of interest.

The term "regulatory sequence" as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term "recombinant host cell" ("expression host cell", "expression host system", "expression system" or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. A "mutant" or "variant" or "derivative" (or equivalent wordings having the same meaning), as used herein, and used in the context of an amino acid or nucleotide sequence, is meant to encompass a subsequent amino acid or nucleotide sequence that has been derived from a previous amino acid or nucleotide sequence, or reference sequence, either naturally or artificially. For purposes of illustration only, a variant or mutant protein of wild type Aequorea GFP (the reference in this case) may have one or more amino acid substitutions, additions, or deletions as compared to said Aequorea wild type GFP amino acid sequence. Specific, but non-limiting, examples of fluorescent protein mutants or variants are provided in Tables 5 and 6 (examples known in the art), as well as in the Detailed Description part hereinafter (as part of the present invention).

A "spectral variant" or "spectral mutant", as used herein, refers to a fluorescent protein to indicate a mutant fluorescent protein that has a different fluorescence characteristic with respect to the corresponding wild type fluorescent protein. For example, CFP, YFP, ECFP, EYFP-V68L/Q69K, and the like, are GFP spectral variants.

A "substitution", as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid. In certain embodiments of the present invention, the substitutions are conservative substitutions. In other embodiments, the substitutions are non-conservative substitutions. Within the context of a polypeptide or protein, conservative and non-conservative amino acid substitutions are known to those of ordinary skill in the art. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

A "deletion", as used herein, is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid. Within the context of a polypeptide or protein, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or a fragment thereof may contain more than one deletion.

An "insertion" or "addition", as used herein, is that change in an amino acid or nucleotide sequence which results from the insertion or addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a parental or reference polypeptide or nucleic acid. "Insertion" or "addition" generally refers to the addition of one or more amino acid residues within an amino acid sequence of a polypeptide, while "addition" can be an insertion or refer to amino acid residues added at an N- or C-terminus, or both termini. Within the context of a protein or a fragment thereof, an insertion or addition is usually of about 1 , about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

The term "antibody" (Ab) refers generally to a polypeptide encoded by an immunoglobulin gene, or antigen-binding fragments thereof, which specifically binds and recognizes an antigen, and is known to the person skilled in the art. A conventional immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The term "antibody" is meant to include whole antibodies, including single- chain whole antibodies, and antigen-binding fragments, which can be produced by digestion with a peptidase or by using recombinant DNA methods. In some embodiments, antigen-binding fragments may be antigen-binding antibody fragments that include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising or consisting of either a VL or VH domain, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to the target antigen. The term "antibodies" is also meant to include heavy chain antibodies, or functional fragments thereof, such as single domain antibodies, for example, nanobodies.

The terms "specifically interact" or "specifically bind" or "specific interaction or binding", as used herein, refers to the ability of one particular polypeptide to preferentially bind to another particular polypeptide, when both polypeptides are present in a homogeneous mixture of different polypeptides. Within the context of the present invention, but without being limitative, the term refers to a peptide that may specifically interact with a protein domain and not, or to a lesser degree, with other (poly)peptides in a mixture of different polypeptides. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable polypeptides in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold). The terms "specifically interact" or "specifically bind" and grammatical equivalents thereof, are used interchangeably herein. The term "affinity", as used herein, refers to the degree to which one particular polypeptide binds to or interacts with another particular polypeptide so as to shift the equilibrium of either polypeptide toward the presence of a complex formed by their binding. Thus, for example, where a peptide and a protein domain are combined in relatively equal concentration, a peptide of high affinity will bind to the available protein domain so as to shift the equilibrium toward high concentration of the resulting complex. Otherwise, a peptide of low affinity will usually not bind to a protein domain, unless one or both of them are present in a high concentration or if their co-recruitment or protein fusion creates a high local concentration. The dissociation constant is commonly used to describe the affinity between two polypeptides, in particular between the peptide and the protein domain. Typically, moderate to strong interactions (moderate to high affinity) imply that the dissociation constant is lower than 10 ^s M. Strong interactions (high affinity) imply a dissociation constant lower than 10^-6 M. Conversely, weak interactions typically imply that the dissociation constant is larger than 10 ^s M.

Detailed description

The strong distance dependence of FRET hampers the wide-spread application of genetically encoded FRET pairs. The present invention provides a strategy for the rational engineering of weak helper interactions that align donor and acceptor fluorophores, leading to robust FRET signals without elaborate optimization of linker sequences or orientations.

A preferred strategy is based on the in s/7/^'co protein design of electrostatically driven heterodimeric encounter complexes and its implementation for the optimization of fluorescent protein pairs for in vitro/in vivo FRET applications. Accordingly, a first aspect of the invention relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a Providing a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements,

b Introducing complementary electrostatic charges in said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins, c. Measuring and comparing the FRET efficiency of said pair of engineered fluorescent proteins relative to the non-engineered pair in a suitable assay,

According to a more specific embodiment, the invention also relates to a method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of: a. Providing a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements,

b. Creating an in silico structural model of a heterodimeric complex of said pair of fluorescent proteins,

c. Introducing and selecting mutations that introduce complementary electrostatic charges within or in the vicinity of the contact interface of said model heterodimeric complex of said pair of fluorescent proteins, resulting in a pair of engineered fluorescent proteins, d. Measuring and comparing the FRET efficiency of said pair of engineered fluorescent proteins relative to the non-engineered pair in a suitable assay,

e. Selecting a pair of engineered fluorescent proteins having increased FRET efficiency relative to the non-engineered pair.

As used herein, the term "fluorescent protein" refers to any protein that can fluoresce when excited with an appropriate electromagnetic radiation. A fluorescent protein may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluorophors, i.e., tryptophan, tyrosine and phenylalanine. A fluorescent protein of the invention or for use in a method of the invention is a protein that derives its fluorescence from autocatalytically forming a chromophore. A fluorescent protein can contain amino acid sequences that are naturally occurring or that have been engineered (i.e., variants or mutants or derivatives, as defined herein). For example, a spectral variant of Aequorea GFP can be derived from the naturally occurring GFP by engineering mutations such as amino acid substitutions into the reference GFP protein. For example, ECFP is a spectral variant of GFP that contains substitutions with respect to GFP. It is to be understood that the term "fluorescent protein", as used herein, also includes variants of fluorescent proteins that have lost actual fluorescence but can still act as FRET acceptors by virtue of a "dark quenching" or "dark absorbing" chromophore. Non- limiting examples are the REACh variants of YFP [39]. Fluorescent proteins are often classified according to their spectral class. Thus, fluorescent proteins may be green fluorescent proteins which fluoresce green, or red fluorescent proteins which fluoresce red, or yellow fluorescent proteins which fluoresce yellow, or cyan fluorescent proteins which fluoresce cyan, or orange fluorescent proteins which fluoresce orange, etc. The term "green fluorescent protein" or "GFP"is used broadly herein to refer to a protein that fluoresces green light, for example, Aequorea GFP. GFPs have been isolated from the jellyfish, Aequorea victoria, the sea pansy, enilla reniformis, and Phialidium gregarium [21, 23]. The term "red fluorescent protein", or "RFP" is used in the broadest sense and specifically covers the Discosoma RFP (DsRed), and red fluorescent proteins from any other species, such as coral and sea anemone, as well as variants thereof, as long as they retain the ability to fluoresce red light[24]. Furthermore, reference is also made to the various spectral variants and mutants that have amino acid sequences that are substantially identical to a reference fluorescent protein. Non-limiting examples of commonly known reference fluorescent proteins include, but are not limited to, A. Victoria GFP (Genebank Accession Number M62654.1), Discosoma RFP (DsRed), (Genebank Accession Number AF168419), amongst others, see [15, 26]; U.S. Patent No. 5,625,048; International application PCT/US95/14692, now published as PCT WO96/23810, each of which is incorporated herein by reference.

Aequorea GFP-related fluorescent proteins include, for example, wild type (native) Aequorea victoria GFP [26], allelic variants thereof, for example, a variant having a Q80R substitution [16]; and spectral variants of GFP such as CFP, YFP, and enhanced and otherwise modified forms thereof (U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079, each of which is incorporated herein by reference), including GFP-related fluorescent proteins having one or more folding mutations, and fragments of the proteins that are fluorescent, for example, an A. victoria GFP from which the two N-terminal amino acid residues have been removed. Several of these fluorescent proteins contain different aromatic amino acids within the central chromophore and fluoresce at a distinctly shorter wavelength than the wild type GFP species. For example, the engineered GFP proteins designated P4 and P4-3 contain, in addition to other mutations, the substitution Y66H; and the engineered GFP proteins designated W2 and W7 contain, in addition to other mutations, Y66W.Similarly, Discosoma RFP (DsRed)-related fluorescent proteins include, for example, wild type (native) Discosoma RFP, allelic variants thereof, and spectral variants thereof, such as mPlum, tdTomato, mStrawberry, DsRed Monomer, mOrange[ll].To illustrate this further, several examples of wild type fluorescent proteins and variants derived thereof are listed in Tables 5 and 6, wherein further details on the properties of each listed protein are provided.

A further class of fluorescent proteins considered here are variants that are derived from other fluorescent proteins like GFP or DsRed or Citrine or mCherry by means of circular permutation. Circular permutation is the fusion of N- and C-terminal of the original protein while simultaneously creating a new N- and C-terminal within the original protein sequence, with the aim to change the orientation of the fluorescent protein within larger protein fusion constructs [17, 19].

Typically, fluorescent proteins are chosen according to the type of application and experiment, essentially based on critical factors such as emission spectra, brightness, photostability, oligomerization, for which guidance is provided in the art, see e.g. [3]. Preferably, the fluorescent proteins as used herein are suitable for Forster resonance energy transfer (FRET), also known as fluorescence energy transfer, or simply, resonance energy transfer (RET), hereinafter further referred to as "FRET". FRET is the nonradiative transfer of excited-state energy from one molecule (the donor) to another nearby molecule (the acceptor) via a long-range dipole-dipole coupling mechanism, and is well described in the art. FRET is usually limited to distances less than ~ 10 nm, and thus provides a sensitive tool for investigating a variety of phenomena that produce changes in molecular proximity. Preferably, the fluorescent proteins as used herein are a pair of fluorescent proteins suitable as a donor and acceptor in FRET measurements, for which guidance is provided in the art, see e.g. [1, 3, 27] as well as in the Detailed Description further herein.

There are several ways to measure or to quantify FRET which are known to the person skilled in the art. Typically, the ratio of donor and acceptor fluorescence is measured (also referred to as ratiometric FRET). Increasingly, instruments and microscopes allow to measure the lifetime of the donor fluorescence, which drops the more energy is transferred to the acceptor. For example, fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. FLIM maps the spatial distribution of probe lifetimes inside living cells, and can accurately measure the shortening of donor lifetimes that result from FRET [29]. In any case, the signal-to-noise ratio depends on the FRET efficiency. The term "FRET efficiency" (£), as used herein, is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event and is known to the person skilled in the art. The FRET efficiency depends on many parameters, including the distance between the donor and the acceptor, the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, the relative orientation of the donor emission dipole moment and the acceptor dipole moment. Preferably, the efficiency of FRET between the donor and acceptor is at least 10%, at least 20%, at least 30%, at least 40%, more preferably around 50%. For intensity-based FRET measurements, the efficiency of FRET is preferably even higher, at least 60%, at least 70%, and even more preferably at least 80%, at least 90%, or higher. One is referred to the Example section for an example on how FRET efficiencies can be calculated. The calculated FRET efficiencies can be compared between two pairs of fluorescent proteins, wherein one pair of fluorescent proteins may have an "increased" or "higher" FRET efficiency or may have a "reduced" or "lower" FRET efficiency relative to the other pair. Preferably, the difference in FRET efficiency is statistically significant.

The rate of energy transfer depends upon the extent of spectral overlap between the donor emission and acceptor absorption spectra, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipole moments, and the distance separating the donor and acceptor molecules. A preferred factor to be considered in choosing the donor and acceptor pair is the efficiency of fluorescence resonance energy transfer between them. The present invention provides guidance on how to improve FRET efficiency. It will be clear that spectral overlap, quantum yield and related spectroscopic properties are a direct consequence of the choice of donor and acceptor protein variant and remain valid for various implementations or applications of the same FRET pair. These were the main criteria that have been used in the past to optimize the efficiency and detectability of FRET between a donor and acceptor molecule. By contrast, distance and orientation could only be influenced by a careful choice of linkers and elaborate design and testing of custom fusion proteins. Therefore, the latter criteria could so far only be optimized on a case-by-case basis for each new FRET sensor or application individually. The present invention offers means to optimize FRET probe distance and orientation largely independent of the molecular and experimental context in which the FRET pair is applied. Distance optimized helper interaction FRET pairs can therefore be used for a variety of applications without or with little further customization.

The term "a pair of engineered fluorescent proteins" or "an engineered pair of fluorescent proteins", as used herein, refers to a pair of fluorescent proteins that has been genetically engineered or modified, according to any of the above described engineering methods; this is in contrast to a non- engineered fluorescent protein pair that has not been genetically engineered or modified according to any of the above described engineering methods, and that is known in the art. It should be clear that a non-engineered pair of fluorescent proteins as described herein is not necessarily restricted to a pair of wild type or reference fluorescent proteins, but may also be, for example, a homolog, a spectral variant or another mutant derived thereof (as defined herein). Preferably, the pair of engineered fluorescent proteins is optimized for higher FRET efficiency as compared to the non-engineered pair. It should be clear that the above described engineering method is meant to improve FRET efficiency by optimizing a pair of fluorescent proteins simultaneously (i.e. donor and acceptor protein), and not the fluorescent proteins individually.

In a preferred embodiment, the above described method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprises the step of introducing complementary electrostatic charges in said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins. The term "complementary electrostatic charges", as used herein, refers to electrostatic interactions between proteins and is mainly determined by the charged residues, in particular at the protein surface. "Introducing complementary electrostatic charges" refers to modifying the number of charged residues of the two proteins in opposite direction, for example, by introducing positively charged amino acids in the first protein and by introducing negatively charged amino acids in the second protein. Preferably, at least one, at least two, at least three, or more amino acid substitutions in at least one of the fluorescent proteins of the pair are introduced. Alternatively, at least four, at least five, or more amino acid substitutions in at least one of the fluorescent proteins of the pair are introduced. Preferably, at least one, at least two, at least three, or more amino acid substitutions are introduced in the two fluorescent proteins of the pair (thus in both fluorescent proteins). Alternatively, at least four, at least five, or more amino acid substitutions are introduced in the two fluorescent proteins of the pair. Preferably, the at least one or more amino acid substitution results in the introduction of at least one positive charge in one fluorescent protein of the pair and the introduction of at least one negative charge in the other fluorescent protein of the pair, or vice versa. For the sake of clarity, it should be clear from the above that "introduction of a positive charge" means introducing a "net positive charge" by introducing one or more positive charge(s) or removing one or more negative charge(s), or combinations thereof resulting in a net positive charge. Similarly, "introduction of a negative charge" means introducing a "net negative charge" by introducing one or more negative charge(s) or removing one or more positive charge(s), or combinations thereof resulting in a net negative charge.

In a preferred embodiment, the above described method of obtaining a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprises the step of creating an in silico structural model of a heterodimeric complex of a pair of fluorescent proteins that is provided and to use this as a template for optimizing said pair for high FRET efficiency. The design or construction of such a hypothetical structural model between the two fluorescent proteins is typically based on available protein structures in the Protein Data Bank (PDB, www.pdb.org; [20]), preferably of at least one of the constituting fluorescent proteins of the pair or a homolog thereof. Preferably, an existing structure of a complex of two identical (homodimer) or homologous fluorescent proteins is used as a "template" homodimer complex. The terms "creating" or "design" or "construction" or semantic derivatives thereof are used interchangeably herein. Typically, structure- and/or sequence- based alignments are used to position donor and acceptor protein structures into the same orientation as the "template" homodimer complex. This is done by making use of computer algorithms known in the art, for example TM-align, which is illustrated in a non-limiting way further herein in the Example section. As used herein, "a homodimeric complex" of two fluorescent proteins means a complex formed by association of two identical fluorescent proteins. The term "a homodimeric interface", as used herein, refers to the residues that are participating in the molecular contacts between the two identical fluorescent proteins of a homodimeric complex. As used herein, "a heterodimeric complex" of two fluorescent proteins means a complex formed by association of two distinct fluorescent proteins. The term a "heterodimeric interface", as used herein, refers to the residues that are participating in the molecular contacts between the two distinct proteins of a heterodimeric complex.

Preferably, the complementary electrostatic charges are introduced within or, more preferably, in the direct vicinity of the modelled heterodimeric contact interface between the two fluorescent proteins of the pair, hereby promoting the weak interaction between both proteins of the pair. The term "contact interface" or "protein-protein interface" or simply "interface", as used herein, refers to the subset of residues of each protein that are in contact with any residue of the other protein. Based on the atomic structure of a protein-protein complex, a residue is commonly defined to be in contact with a residue from the partner protein if at least one atom of the first residue is within 0.5 nm of any atom of the other residue [30]. The term "direct vicinity" or "rim" of an interface, as used herein, is defined as those residues that have at least one backbone or C_p atom within 1.5 nm of a backbone or C_p atom from the partner protein but do not belong to the actual interface (as defined above). That means, all atoms of rim residues are further than 0.5 nm away from all atoms of the partner protein. These atomic distances can preferably be derived from the structure or a structural model of the protein- protein interaction complex. Alternatively, related methods (e.g. docking) can be used to create a target interface which can then serve as a starting point for optimization.

In a further embodiment, the above described method of obtaining a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair further comprises the step of introducing mutations that remove unfavorable contacts within the contact interface of said model heterodimeric complex, causing, for example, steric hindrances (also referred to as "clashes") between the binding partners.

In still another embodiment, the above method may further comprise the step of introducing mutations that disrupt homodimer interfaces without impairing heterodimerisation, as can be predicted from existing homodimeric structures of one or both of the constituting fluorescent proteins of the pair (acceptonacceptor and/or donondonor), or from the homodimeric structure of homologous proteins derived thereof. Preferably, the fluorescent proteins of the pair do not show initial intrinsic interaction, such as fluorescent protein variants that have been made monomeric like mCitrine or mCherry (see also Table 5 and 6). In a specific embodiment, the pair of fluorescent proteins of the method as described herein comprises (m)Citrine (SEQ ID NO: 1), or a variant thereof (as defined hereinbefore), and mCherry (SEQ ID NO: 4), or a variant thereof (as defined hereinbefore). For the sake of clarity, since Citrine is derived from and a variant of GFP, other proteins derived from GFP are automatically also considered variants of Citrine. Likewise, since mCherry is derived from and a variant of Ds ed, other proteins derived from DsRed are also considered variants of mCherry.

In an even more specific embodiment, the pair of fluorescent proteins of the method as described herein comprises: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1, selected from the group consisting of positions 43, 144, 202, 221 and 227, and

b. an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the group consisting of positions 39, 144, 145 and 196.

Preferably, the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: l,and from the group consisting of E39K, E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4. A second aspect of the invention relates to a pair of engineered fluorescent proteins having an increased FRET efficiency relative to a pair of SEQ ID NO: 1 and SEQ ID NO: 4, comprising a donor and an acceptor fluorescent protein, wherein said donor and acceptor fluorescent proteins are chosen from the group consisting of: a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein corresponding to SEQ ID NO: 4;

b. a donor fluorescent protein corresponding to SEQ ID NO: l,and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and

196. c. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, 145 and 196;

Preferably, the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: l,and from the group consisting of E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.

It will be appreciated by one of ordinary skill in the art that the amino acid mutations described herein can be substituted for other amino acid mutations at the specific residue or residues (or a nearby residue or residues) described herein, that achieves the same effect as the novel fluorescent proteins described herein (e.g., increased FRET). It is well within the level of those of ordinary skill in the art to create a pair of fluorescent proteins based on the mutated amino acid residues of the present invention to create additional pairs of fluorescent proteins aside from the ones described herein. Therefore, additional pairs of fluorescent proteins containing an amino acid other than those described herein are encompassed by the instant invention. Further (non-limiting) candidate amino acids are given in Table 7, which is based on the result of the filtering steps described above. For example, a novel pair of fluorescent proteins wherein one or both of the fluorescent proteins are having an amino acid mutation at a position as described herein wherein the position is the site for mutation but the amino acid may differ, is encompassed by the present invention. For example, the mutation at position N144 in SEQ ID NO: 1 is not required to be N144E or N144D, but can be any other mutation as long as the properties of the engineered fluorescent protein pair are not altered or otherwise are enhanced.

For the sake of clarity, the term "variant" as defined hereinbefore, encompasses homologs of the donor or acceptor fluorescent proteins. Homologs of a protein encompass polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question, and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity, (for example, homologous fluorescent proteins derived from one species, e.g. Aequorea GFP, either naturally occurring or made by man). Several different methods are known by those of skill in the art for identifying and defining these functionally homologues sequences, including phylogenetic methods, sequence similarity and hybridization methods. Percentage similarity and identity can be determined electronically. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www/ncbi.nlm. nih.gov/). Preferably, a homolog has a sequence identity at protein level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, preferably at least 90%, even more preferably at least 95%, at least 98%, at least 99%, as measured in a BLASTp or any other equivalent method known in the art.

In a preferred embodiment, the donor and the acceptor fluorescent proteins comprised in the pair are each fused to a molecule of interest, preferably a polypeptide of interest or a target polypeptide, optionally through a linker molecule. A "polypeptide of interest" or a "target protein" or grammatically equivalents thereof, as used herein, can be any polypeptide, including, for example, a sensor protein such as calmoduline, or a cellular polypeptide such as an enzyme, a G-protein, a growth factor receptor, or a transcription factor, amongst others, and can be one or two or more proteins that can interact or associate to form a complex. The engineered donor and acceptor fluorescent protein of the present invention may each be linked to a molecule of interest either directly or indirectly, using any linkage that is stable under the conditions to which the protein- molecule complex is to be exposed. Where the molecule of interest is a polypeptide, a convenient means for linking or fusing a fluorescent protein variant and the molecule is by expressing them as a fusion protein from a recombinant nucleic acid molecule, which comprises a polynucleotide encoding, for example, a fluorescent protein operably linked to a polynucleotide encoding the polypeptide molecule. Preferred "linker molecules" or "linkers" are peptides of 1 to 50 amino acids length and are typically chosen or designed to be unstructured and flexible. These include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins [31]. Examples used herein are (GS)s (SEQ ID NO:31) or (GS)i₀ (SEQ ID NO: 32)or a 50 amino acid randomized sequence of Gly, Serjhr, Gin, Glu (SEQ ID NO: 33). Further examples include, but are not limited to, a protease cleavage site such as Factor Xa cleavage site having the sequence IEG (SEQ ID NO:34), the thrombin cleavage site having the sequence LVPR (SEQ ID NO:35), the enterokinase cleaving site having the sequence DDDDK (SEQ ID NO:36), or the PreScission cleavage site LEVLFQGP (SEQ ID NO: 37). Preferably, the amino acid linker sequence is relatively short, but long enough to allow the contact of enhanced donor and acceptor fluorescent proteins, and does not interfere with the biological activity of the proteins. Note that the terms "linker" and spacer" are used interchangeably herein. Non-limiting examples of suitable linker sequences are also described in the Example section.

Thus, the invention also provides a fusion protein comprising a polypeptide of interest fused to an engineered donor fluorescent protein or to an engineered acceptor fluorescent protein, as described hereinbefore. More specifically, the present invention also encompasses a "bimolecular construct" of two such fusion proteins, wherein one fusion protein comprises a polypeptide of interest fused to an engineered donor fluorescent protein and one other fusion protein comprises a polypeptide of interest fused to an engineered acceptor fluorescent protein. It should be understood that bimolecular constructs refer to two separate fusion proteins. On the other hand, the bimolecular constructs as described herein may be expressed from a single recombinant nucleic acid molecule or from two separate recombinant nucleic acid molecules, as described further herein. Such a bimolecular construct is particularly useful for detection of protein-protein interactions, which, in turn, can serve as indicator of changes in protein signaling, protein modifications or protein localization.

In a more specific embodiment, the invention provides a bimolecular construct comprising: a. a donor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and

b. an acceptor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and wherein said donor and acceptor fluorescent proteins are comprised in the pair of engineered fluorescent proteins as described herein above.

Alternatively, a fusion protein is provided comprising one or more polypeptide(s) of interest, an engineered donor and an acceptor fluorescent protein as described hereinbefore, which is then referred to as a "unimolecular construct" or "tandem construct". The different polypeptides can be fused to each other in any order and either directly or indirectly through one or more linker molecules. Such a unimolecular construct can be particularly useful for the detection of conformational changes or intra-molecular binding within a polypeptide of interest which, in turn, can serve as proxy for changes in cellular signaling, ion concentrations or enzymatic activities. Unimolecular constructs are also used for the detection of peptide cleavage events, in particular, due to protease activities.

Thus, in such an alternative embodiment, the invention envisages a unimolecular protein construct selected from the group comprising a fusion protein construct as follows: a. a donor fluorescent protein - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, or

c. a polypeptide of interest - a donor fluorescent protein - an acceptor fluorescent protein

- a polypeptide of interest, optionally fused through one or more linker molecules, or d. a polypeptide of interest - an acceptor fluorescent protein - a donor fluorescent protein

- a polypeptide of interest, optionally fused through one or more linker molecules, or e. a donor fluorescent protein - a polypeptide of interest - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, f. an acceptor fluorescent protein - a polypeptide of interest - a polypeptide of interest - a donor fluorescent protein, optionally fused through one or more linker molecules, and, wherein said donor and acceptor fluorescent proteins are comprised in the pair of engineered fluorescent proteins as described herein above.

The polypeptides comprised in any of the above described fusion proteins can be linked through peptide bonds. The fusion proteins may be expressed from a recombinant nucleic acid molecule containing a polynucleotide encoding an engineered fluorescent protein of the invention operatively linked to one or more polynucleotides encoding one or more polypeptides of interest.

In a specific embodiment, the present invention also provides an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, such as T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D. In another specific embodiment, the present invention also provides an engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, and 145, such as E39 , E144K, E144R, A145K, A145R, N196K and N196R. A fusion protein comprising a polypeptide of interest fused to the engineered fluorescent protein as described hereinbefore, optionally through a linker molecule, is also envisaged here.

In another embodiment, the invention relates to one, two, or more polynucleotides encoding the engineered donor and/or the acceptor fluorescent proteins, or the engineered fluorescent proteins, or the bimolecular or unimolecular constructs, or the fusion proteins, all as described hereinbefore. Non- limiting examples of polynucleotide sequences encoding engineered fluorescent protein variants and synthetic protein constructs according to the invention, as well as the corresponding amino acid sequences, are listed in Table 8.

The invention further concerns expression vectors containing such polynucleotides and host cells containing such polynucleotides or vectors. The vector generally contains elements required for replication in a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (e.g. Promega, Madison Wl; Stratagene, La Jolla CA; GIBCO/B L, Gaithersburg MD) or can be constructed by one skilled in the art. Where the vector is a viral vector, it can be selected based on its ability to infect one or few specific cell types with relatively high efficiency. For example, the viral vector also can be derived from a virus that infects particular cells of an organism of interest, for example, vertebrate host cells such as mammalian host cells. Viral vectors have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like. The construction of expression vectors and the expression of a polynucleotide in transfected cells involves the use of molecular cloning techniques also well known in the art (see Sambrook et al., In "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press 1989); "Current Protocols in Molecular Biology" (eds., Ausubel et al.; Greene Publishing Associates, Inc., and John Wiley & Sons, Inc. 1990 and supplements).

In a third aspect, an antibody that specifically binds to an engineered fluorescent protein according to the invention is also envisaged here.

A fourth aspect of the invention is drawn to a kit used for making and using a pair of engineered fluorescent proteins according to the invention in laboratory methods or other applicable uses, including, for example, to construct a fluorescent fusion protein comprising a fluorescent protein of the invention that can be expressed in living cells, tissues, and organisms. Preferably, the invention provides kits comprising any of the above described polynucleotides or any of the above described expression vectors. Alternatively, or in addition, the kits can provide any of the above described (pair of) engineered fluorescent proteins or fusion proteins themselves. In addition, kits of the present invention can also include, for example but not limited to, apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, reagents for bacterial cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Also present in the kits may be antibodies specific to the provided proteins. The components of the kit may be packaged in combination or alone in the same or in separate containers, depending on, for example, cross-reactivity or stability, and can also be supplied in solid, liquid, lyophilized, or other applicable form. A container means of the kits may generally include, for example, at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Such a kit may be useful for any of the applications of the present invention as described further herein.

A fifth aspect of the invention relates to the use of any of the engineered fluorescent proteins (whether as a pair or individual, and whether in a bimolecular or unimolecular construct) or any polynucleotide encoding such protein in any method that employs a fluorescent protein. In a preferred embodiment, the engineered fluorescent proteins are useful as a FRET pair for in vitro and/or in vivo FRET-based applications, such as detection of protein-protein interactions, conformational changes of a protein, protease activity, protein modifications, changes in concentrations of metabolites, ions or signaling molecules. In a specific embodiment, the aforementioned FRET-based assay is part of a screening process, for example, in order to discover or characterize compounds, conditions or processes that trigger or disrupt protein-protein interactions or that lead to other changes in the state of cellular or in-vitro systems.

Thus, according to one embodiment, fluorescent protein variants having features of the invention are useful as a FRET pair in a method of identifying a specific interaction of a first molecule and a second molecule, for example a specific interaction between proteins, or between a protein and a nucleic acid, or between nucleic acids. The first and second molecules can be cellular proteins that are being investigated to determine whether the proteins specifically interact, or to confirm such an interaction. Such first and second cellular proteins can be the same, where they are being examined, for example, for an ability to oligomerize, or they can be different where the proteins are being examined as specific binding partners involved, for example, in an intracellular pathway. The first and second molecules also can be a polynucleotide and a polypeptide, for example, a polynucleotide known or to be examined for transcription regulatory element activity and a polypeptide known or being tested for transcription factor activity. For example, the first molecule can comprise a plurality of nucleotide sequences, which can be random or can be variants of a known sequence, that are to be tested for transcription regulatory element activity, and the second molecule can be a transcription factor, such a method being useful for identifying novel transcription regulatory elements having desirable activities. The conditions for such an interaction can be any conditions under which is expected or suspected that the molecules specifically interact. In particular, where the molecules to be examined are cellular molecules, the conditions generally are physiological conditions. As such, the method can be performed in vitro using conditions of buffer, pH, ionic strength, and the like, that mimic physiological conditions, or the method can be performed in a cell or using a cell extract.

Accordingly, in a preferred embodiment, the invention envisages a method of identifying a specific interaction of a first polypeptide of interest and a second polypeptide of interest, the method comprising: a. Contacting the first polypeptide of interest, which is fused to a donor fluorescent protein of a pair of engineered fluorescent proteins according to the invention, and the second polypeptide of interest, which is fused to the corresponding acceptor fluorescent protein of the pair of engineered fluorescent proteins according to the invention, under conditions that allow a specific interaction of the first and the second polypeptide of interest,

b. Exciting the donor, and

According to specific embodiments of the above method, the first polypeptide is a first cellular protein or fragment thereof and the second polypeptide is a second cellular protein or fragment thereof,.

The use of the fluorescent protein variants of the invention for such a purpose provides a substantial advantage in that (1) the FRET signal is increased over noise and background, (2) FRET signals can be obtained without optimizing the distance and orientation between the interacting proteins of interest and the donor and acceptor probes which, (3) allows to use standardized long (rather than customized short) molecular linkers between proteins of interest and fluorescent probes, which (4) diminishes the risk of artifacts or modification of the biological activity of interest.

The above processes can be miniaturized and automated to enable screening many thousands of molecules in a high throughput format.

In another embodiment, the engineered fluorescent proteins are useful as a FRET pair to detect cleavage of a substrate having the donor and acceptor coupled to the substrate on opposite sides of the cleavage site. Upon cleavage of the substrate, the donor/acceptor pair physically separate, eliminating FRET. Such an assay can be performed, for example, by contacting the substrate with a sample, and determining a qualitative or quantitative change in FRET. A fluorescent protein variant donor/acceptor pair also can be part of a fusion protein coupled by a peptide having a proteolytic cleavage site. In other embodiments, useful applications include F ET-based sensors for protein kinase and phosphatase activities or indicators for small ions and molecules such as Ca²⁺, Zn²⁺, cyclic 3', 5'- adenosine monophosphate, and cyclic 3', 5' -guanosine monophosphate.

Further, the fluorescent protein variants are useful as fluorescent markers in the many ways fluorescent markers already are used, including, for example, coupling fluorescent protein variants to antibodies, polynucleotides or other receptors for use in detection assays such as immunoassays or hybridization assays, or to track movement of proteins in cells. Examples wherein the fluorescent protein variants can be used as labeling substance, include, without the purpose of being limitative, biological and/or medicinal imaging, fluorescent microscopy, FRET-based assays or screening or imaging (in vitro, in cells or in vivo).

Fluorescence in a sample generally is measured using a fluorimeter and methods of performing assays on fluorescent materials are well known in the art (see, for example, Lakowicz, "Principles of Fluorescence Spectroscopy" (Plenum Press 1983); Herman, "Resonance energy transfer microscopy" In "Fluorescence Microscopy of Living Cells in Culture" Part B, Meth. Cell Biol. 30:219-243 (ed. Taylor and Wang; Academic Press 1989); Turro, "Modern Molecular Photochemistry" (Benjamin/ Cummings Publ. CoJ, jfric. 1978), pp. 296-361, each of which is incorporated herein by reference).

The sample to be examined can be any sample, including a biological sample, an environmental sample, or any other sample for which it is desired to determine whether a particular molecule is present therein. Preferably, the sample includes a cell or an extract thereof. The cell can be obtained from a vertebrate, including a mammal such as a human, or from an invertebrate, and can be a cell from a plant or an animal. The cell can be obtained from a culture of such cells, for example, a cell line, or can be isolated from an organism. As such, the cell can be contained in a tissue sample, which can be obtained from an organism by any means commonly used to obtain a tissue sample, for example, by biopsy of a human. Where the method is performed using an intact living cell or a freshly isolated tissue or organ sample, the presence of a molecule of interest in living cells can be identified, thus providing a means to determine, for example, the intracellular compartmentalization of the molecule.

The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein. EXAMPLES

EXAMPLE 1. Model protein-protein interaction FRET sensor Target

Citrine [10] and mCherry [11] are among the most widely used fluorescent protein (FP) variants. The yellow FP Citrine [10] is derived from Aequorea victoria GFP. It is assumed to retain the weak homodimerization tendency of GFP but can be converted into a strict monomer (mCitrine) by the interface-breaking mutation A206K [5]. mCherry [11] is a monomeric and improved variant of Discosoma red fluorescent protein. As the two fluorophores originate from different species (jelly fish and coral), they show no intrinsic interaction. (m)Citrine / mCherry are an excellent long-wavelength FRET pair combining good spectral separation and single exponential donor decay kinetics with among the longest Forster distance reported for any genetically encoded pair [12].

Implementation

We used the chemically induced protein-protein interaction between FRB and FKBP12(T2089L) [13, 14] as a test system for conventional and enhanced FRET probes. As outlined in Figure lb, addition of rapamycin triggers a high-affinity binding between the two domains yet there is no interaction in absence of the drug [14]. This allowed us to measure FRET signals both in bound (active, induced) and unbound state.

FRET donor constructs all shared the same primary structure architecture and were composed (from N- to C-terminal) of: (1) a FKBP12 domain (SEQ ID NO: 11) preceded by a Thr-Gly spacer, (2) a 10 (SEQ ID NO: 31) or 20 amino acid linker (SEQ ID NO: 32) consisting of 5 or 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 14 or 24 amino acids), (3) the mCitrine reference or modified sequence followed by a Ser-Gly spacer (see Table 8), (4) a recognition and cleavage site for the PreScission protease (SEQ ID NO: 37) followed by a Thr-Gly spacer, (5) a hexa -Histidine tag for purification purposes followed by a Thr-Gly spacer and STOP codon. FRET acceptor constructs all shared the same primary structure architecture and were composed (from N- to C-terminal) of: (1) a FRB domain preceded by a Thr-Gly spacer, (2) a 10 or 20 amino acid linker consisting of 5 or 10 repeats of a Gly-Ser dipeptide and preceded and followed by a Thr-Gly spacer (amounting to an overall linker length of 14 or 24 amino acids), (3) the mCherry reference or modified sequence followed by a Ser-Gly or a Gly-Ser spacer, (4) a recognition and cleavage site for the PreScission protease followed by a Thr-Gly spacer, (5) a hexa-Histidine tag for purification purposes followed by a Thr-Gly spacer and STOP codon. Table 3 lists all proteins constructed for this study.

We improved our previous protocol for in vitro FRET measurements[14]: (1) Purification by gel filtration was crucial for consistent results. (2) Extinction coefficients (see Table 4) were determined by comparison with absorbance at 280nm and averaged over various protein constructs containing (m)Citrine or mCherry and are somewhat different from published values [3]. (3) To account for inevitable inaccuracies in protein concentrations, we ensured full donor or acceptor occupancy by providing acceptor protein in excess during the measurement of donor-based FRET and, vice-versa, an excess of donor for sensitized emission measurements. In vitro FRET efficiencies measured with the modified protocol are typically reproduced to within 1% E also between different preparations of equivalent proteins. A good agreement between donor- and acceptor-based FRET measurements (Figure 6) indicates that (m)Citrine and mCherry populations were generally homogeneous and intact.

EXAMPLE 2. FRET enhancement by electrostatic helper interaction

The anti-parallel homodimer structure of Aequorea GFP served as a template for the design of a weak Citrine / mCherry heterodimer. Structures of the two proteins were brought into the same orientation and side chain refinement left no major steric clashes (Figure 2a). However, the monomerizing mutation A206K[5] was expected to also hinder any heterodimerization and was therefore reverted. Instead a Citrine/Citrine homodimer model was serving as "negative" target for design against homodimerization. DsRed oligomerization interfaces are very different and an accidental (re)introduction of mCherry homodimerization was thus not likely. Rather than designing an intricately optimized, and potentially too strong, new interface, we tried to stabilize the heterodimer by complementary electrostatic charges placed in the immediate surroundings, but not inside, of the hypothetical interface (Figure 2a). The FoldX protein design algorithm version 3.b4 [36, 37] was used for an exhaustive in silico screen through several thousand combinations of up to five simultaneous mutations on both sides of the synthetic interface. Sets of mutations were selected for their de-stabilization of Citrine homodimers and then ranked by predicted change in free energy of binding promoting heterodimerization. The top- and another high-ranking pair are shown in Figure 2b and described in Table 1. Four additional negative charges on the Citrine and six positive charges on the mCherry side were predicted to yield a moderate gain of binding free energy. The same Citrine mutant could be paired with a related mCherry variant containing only five extra positive charges. Separate calculations later suggested that these mutations might, in fact, remain compatible with mCitrine's A206K mutation (Table 1). The two heterodimer candidates as well as variants with A206K were thus subjected to experimental testing by size exclusion chromatography. No indication of homodimerization for any of the (m)Citrine or mCherry variants, including conventional Citrine 206A was found.

FRET results

We tested the enhanced FRET pair by modifying the model interaction sensor described above; For donor constructs, the (m)Citrine sequence (3 above) was replaced by the modified (m)Citrine 4- followed by a Thr-Gly spacer. For acceptor constructs, the mCherry sequence (3 above) was replaced by the modified mCherry 5+ or the modified mCherry 6+ followed by a Thr-Gly spacer.

Figure 3 compares FRET efficiencies of different conventional and electrostatically enhanced protein pairs. Example fluorescence spectra and lifetime traces are given in Figure 4. Prolongation of the flexible linker between binding and reporter domains leads to the expected drop in FRET efficiency, from 29.9±0.3 % to 23.7±0.6 %. On the other hand, the conventional FRET signal is not influenced by the monomer-enforcing Citrine A206K mutation. The implementation of the top-ranking electrostatic interaction (4-/6+) increases FRET efficiencies to 49.4±0.8 % and 45.6±0.6 % for short and longer linker, respectively. In-line with calculations, the lower-ranking variant (4-/5+) yields slightly lower signals albeit still much improved over the conventional FRET pair. Also the monomer-enforcing Citrine mutation A206K, situated in the center of the interface, results in similarly small but significant reductions of all enhanced FRET efficiencies.

The FRET efficiency between unmodified (m)Citrine and mCherry remains constant over a wide range of ionic strength (Figure 7), indicating that the two probes are freely tumbling without noticeable electrostatic attraction or repulsion. By contrast, the electrostatic helper interactions are strongly affected. As expected, de-screening with high salt concentration (0.5M NaCI) nearly abolishes any enhancement of FRET signals. Conversely, non-physiologically low salt concentrations (40μΜ NaCI) boost FRET efficiency to 62+1% and eliminate the difference between stronger and weaker interface.

EXAMPLE 3. Specificity and background of enhanced FRET probes

None of the various engineered electrostatic interfaces did cause any significant background signal (FRET signal in unbound state) at the standard 0.5μΜ protein concentrations of our in vitro experiments. Neither was any background observed at 10-fold higher concentrations (Figure 5a).

With regard to specificity, as shown in Figure 5b,the modification of mCherry alone (6+), paired with wt Citrine, already enhanced FRET signals from 23.7±0.6 % to 37.5±0.5%, compared to 45.6±0.6 % for the complete pair of enhanced FPs. The complementary modification of Citrine (4-) was not showing any enhanced FRET by itself.

EXAMPLE 4. Lifetime measurements

Table 2 compares FRET efficiencies of the most important FRET pairs characterized with various protocols in bulk samples as well as in an in vitro microscopy setup. Absolute FRET efficiencies are consistent across a variety of measurement methods. In particular, efficiencies obtained from the decrease of amplitude-weighted average lifetimes (x_amp) confirm the results of intensity-based experiments (Figure 4). Donor fluorescence decay remained monoexponential before the induction of protein-protein interactions, testifying to the absence of background FRET. Post induction, the enhancement of FRET was evident from the shortening of average lifetimes. Interestingly, a simple double exponential was only a poor model for the excited state time course of both conventional and enhanced FRET pairs, indicating substantial freedom for the sampling of different relative orientations and distances. A stretched exponential model [18] accounts for the dynamic averaging over disordered ensembles and resulted in quantitative agreement between intensity and life-time-based FRET efficiencies.

METHODS TO THE EXAMPLES METHODS

In-silico interface design

Structures of Citrine (PDB code 1HUY) and mCherry (PDB code 2H5Q) were superimposed on the anti- parallel homodimer of wild type GFP (PDB code 1W7S, chains A and D) using the TM-align program (wrapped by the Biskit Python library). We then searched this heterodimer model for surface positions that were (i) amenable to mutation, (ii) outside the core interface but (iii) close enough for intermolecular electrostatic interactions between charged side chains: (i) In an initial set of FoldX calculations, which was performed on Citrine and mCherry individually, all surface residues were mutated to Glu and Asp (Citrine) or Lys and Arg (mCherry), respectively. Only positions that could be mutated without affecting protein stability (ΔΔ6 <0.5 kcal/mol) were considered further, (ii) A residue- residue contact matrix was then calculated based on backbone and C atoms only and filtered for "rim" positions that were located between 0.5 and 1.5 nm distance from a backbone or C atom of the other protein, (iii) Among these, Citrine and mCherry "rim" residue positions that were within 1.5 nm of each other were then compiled into a final list of 98 candidate intermolecular residue-residue contacts. Each candidate contact was subjected to four FoldX calculations testing all combinations of mutations into Glu and Asp on the Citrine and into Arg and Lys on the mCherry side. Individual mutation pairs predicted to stabilize the complex(AAG <-0.2 kcal/mol) and to de-stabilize the Citrine homodimer (AAG >0.0 kcal/mol) were selected for a final combinatorial screen. However, "rim" mutations rarely had strong effects on homodimer stability. Yet, the classic GFP homodimer breaking mutation A206K was expected to be also detrimental for any heterodimeric complex. "Core" interface positions were thus screened for more conservative homodimer-destabilizing mutations that could substitute for A206K. All contact-based and selected homodimer-breaking mutations were then merged into a final combinatorial FoldX screen with up to 5 simultaneous mutations each on the Citrine and mCherry side. Again, the same mutation sets were also evaluated in the context of Citrine/Citrine homodimer models. Mutation sets that were de-stabilizing Citrine homodimers by at least 2 kcal/mol were ranked by their predicted change in free energy of heterodimeric binding and two pairs were selected for experimental testing: (1) the overall best ranking pair (Citrine 4- / mCherry 6+) and (2) a pair ranking #5 but involving the same Citrine mutations (Citrine4- / mCherry 5+). No further variants were tested. Electrostatic potentials

Electrostatic potentials in Figure 2were calculated by numeric solution of the Poisson-Boltzmann equation with the DelPhi v program [38]. Donor and acceptor structures were refined in complex by FoldX, separated, hydrogens added with Chimera[25], atom names adapted to Amber conventions and histidine residues renamed according to protonation state (Biskit). DelPhi calculations were performed using 12 points per nm grid spacing, 60% maximum filling of linear lattice dimensions, 0.14 nm probe radius, dielectric constants of 80 (solvent) and 4 (protein interior), Amber partial charges and 0.15 M ionic strength with 0.2 nm ionic radius. Potential maps and surfaces were visualized with the UCSF Chimera package[25].

For the calculation of electrostatic free energies in Table 1 a workflow for DelPhi calculations was refined and automated in Python modules which are available as open source from the BisKit project web site (http://biskit.pasteur.fr). In brief, the workflow consists of (1) identification and capping of premature chain ends or chain breaks with NME or ACE residues, (2) adding of hydrogens and optimization of protonation states and hydrogen bonding networks with the program Reduce [32], (3) matching and assignment of all-atom partial atomic charges from the Amber FF03 forcefield [33], (4) execution of DelPhi calculations with parameters as described above, except that grid density was increased to 24 points per nm. (5) Using identical grid dimensions, calculations were repeated 6 times for protein "A", "B" and complex "AB" with and without contribution of solvent ions, respectively. Electrostatic binding free energies were calculated according to the energy partitioning method:

Where:

withG^coul being the Coulomb energy, AG^solv being the solvation energy and AG^ions being the energetic contribution from ionic charges in the solvent. Note, for better comparison with foldX values, Table 1 reports AAG, that means changes in AG with respect to the heterodimer model built from unmodified Citrine and mCherry.

Construct design and cloning

Initial protein-coding constructs were obtained by gene synthesis from DNA 2.0 (CA, USA) and delivered in expression plasmid pJExpress411, codon-optimized for E.coli. These constructs were then modified by In-Fusion (Clonetech) or SLIC 26 recombining of PCR products as well as site-directed mutagenesis. Unless noted otherwise, all PCR reactions were performed with Phusion Hotstart polymerase (NEB). (1) Fk-10-Cit constructs33 and 34 were generated by a three-way In-Fusion reaction that simultaneously replaced the Strep tag and 206Kof original versions (not shown) by His tag and 206A. (2) A modified SLIC protocol was used to replace short by longer peptide linkers between binding and FP domain (constructs 41 to 45). Briefly, complementary 87 and 84 nt oligonucleotides encoding 20aa [GS]x linkers were annealed such that there remained two 21 or 18 nt 5'single-stranded overhangs. Synthetic linker and PCR-generated vector fragments (opened to exclude the short linker region) were recombined by SLIC but the linker with its pre-existing overhangs was not subjected to the exonuclease activity of T4 DNA polymerase. (3) Citrine variants with 206K (constructs 47, 80, 81) were generated by site-directed mutagenesis with Pfu-Turbo polymerase(NEB) from constructs 45, 33, 34, respectively using complementary primers. (4) Fk-20-Cit(K,wt) construct 46was generated by three- way SLIC from construct 33 as described but replacing linker and 206A simultaneously. All constructs were verified by DNA sequencing. Protein expression and purification

Expression plasmids were transformed into f. coli BL21(DE3) (Invitrogen). Starter cultures (LB, 50μg/ml kanamycin) were inoculated from single colonies, grown over night at 37°C and then used for 1:100 inoculation of 0.5 I production cultures (2xTY, 50 μg/ml kanamycin). Production cultures were grown shaking to an O.D. of around 0.6, induced with 0.5 mM IPTG and incubated over night at 20°C. Cells were harvested by centrifugation for 15 min at 6000 g and 4°C , washed once in 15 ml PBS, weighed and stored at -80°C . Pellets were resuspended in 5 ml/(g pellet) BugBuster lysis buffer (Novagen), supplemented with Complete protease inhibitor(Roche) at 1 tablet/50 ml. The lysis mix was incubated for 20 min slowly shaking at room temperature. Cell debris was removed by 5 min centrifugation at 1500 g at 4°C , followed by 30 min centrifugation at 20,000 g and4°C to remove insoluble protein. The supernatant was mixed with 4 ml Ni-NTA Agarose resin (Qiagen, washed twice), diluted to 40 ml with binding buffer (25 mM Tris-Hcl, 20 mM imidazole, 0.5 M NaCI, 10% glycerol, pH7.4) and incubated rotating for 30 min at 4°C . The resin was washed (1 min centrifugation at 2000 g) twice with40 ml washing buffer (25 mM Tris-Hcl, 40 mM imidazole, 1 M NaCI, 10% glycerol, 0.1% Tween 20, pH 7.4),transferred to gravity flow columns (BioRad), settled with 30 ml binding buffer and protein was then eluted by gravity flow with 2 x 1 ml elution buffer (25 mM Tris-Hcl, 0.5 M imidazole, 0.5 M NaCI, 10% glycerol, pH 7.4). Samples were clarified again and then subjected to gel filtration on a Superdex75 16/300 column (GE Healthcare)using modified HBSP+ running buffer (10 mM HEPES, 300 mM NaCI, 50 μΜ EDTA, 0.05% P20, 10% glycerol, pH 7.4). Peak fractions were pooled and concentrated into HBSP+ storage buffer (as above but 150 mM NaCI andl mM DTT) with centrifugal spin concentrators (GE Healthcare) with 10 kD size exclusion limit. Protein concentrations were determined from absorbance at 280 nm of native protein samples using sequence-based extinction coefficients [27] as calculated by the ProtParam server [28]. ln-vitro FRET

Measurements were performed in 150 μΙ volumes in black flat-bottom 96-well plates with 0.3 μΜ final donor and 0.5 μΜ final acceptor concentration (for donor FRET efficiency) or, vice versa, 0.5 μΜ final donor and 0.3 μΜ acceptor concentration (for sensitized emission) in HBSP+ buffer (see above) at pH 7.4and 25 to 27°C . All proteins were pre-diluted to 15 μΜ and then diluted to working solutions of 0.45 μΜ donor9and 1.5 μΜ acceptor (donor FRET) or 0.75 μΜ donor and 0.9 μΜ acceptor (sensitized emission). Six replicas each of three samples were prepared for each protein pair on a single plate: (D) 100 μΙ donor + 50 μΙ buffer, (A)100 μΙ buffer + 50 μΙ acceptor, (AD) 100 μΙ donor + 50 μΙ acceptor. The FRB / FKBP12 interaction was triggered by adding 2 μΙ 112.5 μΜ rapamycin to a final concentration of 1.5 μΜ. After 15 s shaking, donor fluorescence was measured at 495 nm excitation and 530 nm emission wavelengths. Sensitized emission was determined at 516 nm(Citrine) excitation and 610 nm (mCherry) emission. Measurements were performed before and after addition of rapamycin and, for control of full binding, repeated after an additional dose of 2 μΙ rapamycin. The donor-based FRET efficiency E was calculated as: / 530 530\

whereF _Dis the fluorescence intensity of the donor-only sample (D), and F _AD s the intensity of the donor+acceptor sample (AD) corrected for (in practice negligible) acceptor "bleed-through" F⁵³⁰ _A. For control purposes, efficiency of FRET was also calculated from measurements before and after addition of rapamycin:

Efficiency of FRET was also calculated, indirectly, from the sensitized emission of the acceptor after donor excitation at 516 nm:

Equation 3 still needs correction for F⁶¹⁰^ the (non-negligible) donor fluorescence at Cherry emission wavelengths. F₆io°is determined from the donor-only measurements (D) but is subject to FRET-based quenching in the mixed (AD) sample:

Where \s the correction factor for apparent donor quenching which was determined

from 495 nm excitation / 516 nm emission measurements in the same experiment. Note, is

derived from the apparent donor-based FRET efficiency E_app which was determined according to equation 1 but is lower than E_D above, owing to the fact that the donor was not fully occupied during the sensitized emission measurements. All fluorescent intensities F were averages of five to six replicates. Overall standard deviations for E were determined by error propagation. Standard deviations reported for E_A do not include the error arising from the determination of extinction coefficients ε⁵¹⁶ _Aand ε⁵¹⁶ _D(Table 4). Owing to these additional uncertainties, E _A is unlikely to perfectly agree with E_D but serves as a consistency check. Large deviations of E_A typically indicate errors in protein extinction coefficients and concentrations but can also hint at systematic defects in binding or fluorescent domains. ln-vitro FLIM

FLIM (fluorescence lifetime imaging)-F ET was measured by time-correlated single-photon counting (TCSPC) with an inverted multiphoton laser scanning microscope (Leica TCS SP5) using a 63x water immersion N.A 1.2 Plan-Apochromat objective, and equipped with a single molecule detection platform and single-photon counting electronics (PicoHarp 300) from PicoQuant GmbH (Berlin). Donor (mCitrine) two-photon excitation was performed at 950nm from a Mai Tai Ti:Sapphire laser (Spectra Physics) with a repetition rate of 80 MHz. Photons were detected by a SPAD set up (PicoQuant). A fluorescence bandpass filter (500-550 nm) limited the detection to the donor fluorescence only.

In vitro FLIM measurements were performed in 500μΙ volumes in Lab-Tek 8-well coverglass dishes with 0.3 μΜ final donor and 0.5 μΜ final acceptor concentration in HBSP+ buffer at pH 7.4. Three samples were prepared for each protein pair on a single dish: (D) 250μΙ donor + 250μΙ buffer, (DA) 250μΙ donor + 250μΙ acceptor, (DA) 250μΙ donor + 250μΙ acceptor. The FRB - FKBP12 interaction was triggered by adding 5μΙ rapamycin to a final concentration of 1.5μΜ. Donor fluorescence was measured before and after addition of rapamycin. Fluorescent decay curves were analyzed in IGOR Pro (WaveMetrics, Portland, Oregon). Mean FRET efficiency values, E, were calculated from:

vjherer_DA is the amplitude-weighted mean fluorescence lifetime of the donor (mCitrine) in the presence of both acceptor (mCherry) and rapamycin. T_D is the mean fluorescence lifetime of the donor (mCitrine) in the presence of acceptor (mCherry) without rapamycin.

For non-FRET conditions, T_Dof the donor in the presence of the acceptor but without rapamycin was calculated from a mono-exponential fit to the fluorescence lifetime decays. Under FRET conditions, experimental decay curves were fit to a stretched bi-exponential model [18]. The noninteracting protein's lifetime was fixed to r_D and the value of r_DA and stretching factor β were estimated. Table 1: ln-silico interface designs

Table 2: Selected FRET efficiency results

Table 3: Synthetic proteins constructed in this study.

Table 4: Extinction coefficients in M^~1cm

Table 5: Properties of common fluorescent protein variants per spectral class (1)

¹ ' References: [3, 34, 35]; US patent application US2006/0275827; www.eyrogen.com; www.clontech.com

Table 6: Mutations of common fluorescent protein variants[3, 11] )

such as K26R, Q80R, N146H, H231L, etc. variants

Many GFP variants contain V inserted after Metl so that the mRNA should contain an ideal translational start sequence. We number such a V as la to preserve wild-type numbering for the rest of the sequence.

Table 7: Surface-exposed Citrine/Cherry interface rim positions amenable to mutation

Table 8: Overview amino acid and nucleotide sequences used in this study.

Non-limiting examples of reference fluorescent proteins, engineered fluorescent protein variants, test protein-protein interaction and synthetic protein constructs

¹ 'For nomenclature, see also Table 1

⁽²⁾ For nomenclature, see also Table 3

⁽³⁾ X can be amino acid A or K

⁽⁴⁾ Nucleotide sequences of synthetic protein constructs can be easily derived by combining the nucleotide sequences as provided in SEQ ID NOs: 20-30. REFERENCES

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Claims

CLAIMS 1. A pair of engineered fluorescent proteins having an increased FRET efficiency relative to a pair of SEQ ID NO: 1 and SEQ ID NO: 4, comprising a donor and an acceptor fluorescent protein, wherein said donor and acceptor fluorescent proteins are chosen from the group consisting of:

a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, and an acceptor fluorescent protein corresponding to SEQ ID NO: 4;

2. The pair according to claim 1 wherein the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: 1, and from the group consisting of E39R, E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.

3. The pair according to any of claims 1 or 2 wherein the donor and the acceptor fluorescent proteins are each fused to a polypeptide of interest, optionally through a linker molecule.

4. A bimolecular construct comprising:

a. a donor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and

b. an acceptor fluorescent protein fused to a polypeptide of interest, optionally through one or more linker molecules, and wherein said donor and acceptor fluorescent proteins are as defined in any of claims 1 or 2.

5. A unimolecular protein construct selected from the group comprising a fusion protein construct as follows: a. a donor fluorescent protein - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, or

- a polypeptide of interest, optionally fused through one or more linker molecules, or e. a donor fluorescent protein - a polypeptide of interest - a polypeptide of interest - an acceptor fluorescent protein, optionally fused through one or more linker molecules, f. an acceptor fluorescent protein - a polypeptide of interest - a polypeptide of interest - a donor fluorescent protein, optionally fused through one or more linker molecules, and, wherein said donor and acceptor fluorescent proteins are as defined in any of claims l or 2.

6. An engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1 selected from the group consisting of positions 43, 144, 202, 221 and 227, such as T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D.

7. An engineered fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 4 selected from the group consisting of positions 39, 144, and 145, such as E39 , E144K, E144R, A145K, A145R, N196K and N196R.

8. A fusion protein comprising an engineered fluorescent protein as defined in claims 6 or 7 fused to a polypeptide of interest, optionally through one or more linker molecules.

9. A polynucleotide encoding the donor and/or the acceptor fluorescent proteins as defined in claims 1 or 2, or the engineered fluorescent proteins as defined in claims 6 or 7, or the bimolecular construct as defined in claim 4, or the unimolecular protein construct as defined in claim 5, or the fusion protein as defined in claim 8.

10. An expression vector comprising suitable expression control sequences operably linked to any of the polynucleotides of claim 9.

11. A host cell comprising any of the polynucleotides according to claim 9 or any of the expression vectors according to claim 10.

12. A kit comprising any of the polynucleotides according to claim 9 or any of the expression vectors according to claim 10.

13. An antibody that specifically binds to the engineered fluorescent protein according to any of claims 6 or 7.

14. Use of the pair according to any of claims 1 to 3, or the protein constructs according to any of claims 4 or 5, or the engineered fluorescent proteins according to any of claims 6 or 7, or the fusion protein according to claim 8, or any of the polynucleotides according to claim 9 or any of the expression vectors according to claim 10, for in vitro and/or in vivo FRET-based applications.

15. A method of identifying a specific interaction of a first polypeptide of interest and a second polypeptide of interest, the method comprising:

a. Contacting the first polypeptide of interest, which is fused to a donor fluorescent protein of a pair of engineered fluorescent proteins as defined in claims 1 or 2, and the second polypeptide of interest, which is fused to the corresponding acceptor fluorescent protein of the pair of engineered fluorescent proteins as defined in claims 1 or 2, under conditions that allow a specific interaction of the first and the second polypeptide of interest,

b. Exciting the donor, and

16. A method of producing a pair of engineered fluorescent proteins having an increased FRET efficiency relative to the non-engineered pair comprising the steps of:

a. Providing a pair of fluorescent proteins suitable as a donor and acceptor for FRET measurements,

c. Introducing and selecting mutations that introduce complementary electrostatic charges within or in the vicinity of the contact interface of said model heterodimeric complex of said pair of fluorescent proteins resulting in a pair of engineered fluorescent proteins. d. Measuring and comparing the FRET efficiency of said pair of engineered fluorescent proteins relative to the non-engineered pair in a suitable assay,

17. The method according to claim 16 further comprising the steps of introducing mutations that remove unfavorable contacts within the contact interface of said model heterodimeric complex and/or introducing mutations that disrupt homodimer interfaces without impairing heterodimerisation.

18. The method according to any of claims 16 to 17 wherein the fluorescent proteins of the pair do not show initial intrinsic interaction.

19. The method according to any of claims 16 to 18 wherein said introducing of complementary electrostatic charges is further defined as introducing at least one amino acid substitution in at least one of the fluorescent proteins of the pair.

20. The method according to any of claims 16 to 19 wherein said introducing of complementary electrostatic charges is further defined as introducing at least one positive charge in one fluorescent protein and at least one negative charge in the other fluorescent protein.

21. The method according to any of claims 16 to 20 wherein said pair of fluorescent proteins comprises (m)Citrine (SEQ ID NO: 1), or a variant thereof, and mCherry (SEQ ID NO: 4), or a variant thereof.

22. The method according to any of claims 16 to 21 wherein said pair of engineered fluorescent proteins comprises:

a. a donor fluorescent protein comprising at least one amino acid substitution at a position corresponding to a position in SEQ ID NO: 1, selected from the group consisting of positions 43, 144, 202, 221 and 227, and

23. The method according to claim 22 wherein the at least one amino acid substitution is selected from the group consisting of T43E, T43D, N144E, N144D, S202E, S202D, L221A, A227E and A227D in SEQ ID NO: l,and from the group consisting of E39K, E39 , E144K, E144R, A145K, A145R, N196K and N196R in SEQ ID NO: 4.