WO1989009233A1

WO1989009233A1 - Molecular sticks for controlling protein conformation

Info

Publication number: WO1989009233A1
Application number: PCT/US1989/001195
Authority: WO
Inventors: Lawrence M. Kauvar
Original assignee: Terrapin Technologies, Inc.
Priority date: 1988-03-24
Filing date: 1989-03-23
Publication date: 1989-10-05
Also published as: AU3439589A

Abstract

Methods of stabilizing the three-dimensional conformations of proteins using "molecular sticks" to bridge and constrain tertiary protein structures are disclosed. These "molecular sticks" contain rigid multimeric spacer portions of various lengths, which are terminated on each end by chemical groups capable of covalent linkages to other designated chemical groups on different segments of the protein or peptide to be constrained. Proteins or peptides with stabilized tertiary structures are useful in diagnosis and therapy, especially under conditions where unstabilized proteins would be denatured.

Description

MO ECULAR STICKS FOR CONTROLLING PROTEIN CONFORMATION

Technical Field

The invention relates to protein synthesis and control of conformation. In particular, it concerns link¬ ing distal regions on a target peptide with a rigid molecular stick to restrain the three-dimensional conformation of the protein in solution.

Background Art

It has long been understood that proteins of amino acid sequence greater even than about two residues assume preferred conformations in solution depending on^' the ionic strength, pH, and redox conditions. In a general way, the classical hypothesis that the primary structure predetermines the three-dimensional conformation by preferential hydrogen bond formation in alpha-helices, burial of hydrσphobic residues in the portion of the molecule away from an aqueous medium, the display of highly polar and/or ionized residues at the surface of the molecule in aqueous medium, and stabilization of this conformation by formation of intrachain (and sometimes interchain) disulfide bonds has been shown to have some validity. However, this model is somewhat too simple in that, for one thing, the preferred conformation represents a statistical result, so that at a given moment not all molecules of the protein necessarily assume this preferred conformation. Also variation in the conditions in which the protei finds itself may favor alternate conformations which may or may not be corrected or correctable if and when the conditions are again returned to physiological. Indeed, the problem of protein "denaturation" has long been recognized as exemplifying precisely this latter tendency.

The construction of immunologically reactive and specific proteins and peptides or proteins and peptides reactive with receptor is, in particular _r complicated by this variability in three-dimensional conformations.

Since immunoreactivity or reaction with a receptor is a direct function of the contours, both spatial and charge contours, of the molecule, control only of primary structure of a hapten or ligand does not necessarily suf- fice to confer the deeired characteristics. This problem is, of course, also important in connection with any bio¬ logically active molecule, including those which are intended to fit certain biological receptors, to display certain enzymic activities, and so forth. There have been a number of attempts to control three-dimensional^*conformation in peptides of defined primary structure by providing, for example, cyclic residues which force turns in the chain, and also by providing non-labile alternatives for hydrogen bonding to effect a rigid alpha helix (Arrhenius, T., et al, Research Institute of Scripps Clinic, Scientific Report (1986-7) 1_ :167). These methods are somewhat limited in that they operate only over a relatively short range of chain lengths, and do not secure the entire macromolecule into the desired three-dimensional state.

The method of the invention, however, permits the securing of either close or distant points along the primary sequence to constrain the protein to the desired form- By appropriate choice of chemistries _r the method and compositions of the invention can be adapted to any desired protein.

Disclosure of the Invention The invention provides methods and compositions which are useful in providing desired three-dimensional conformations of proteins and peptides. It employs rigid molecular sticks having means of attachment at either end of the rigid stick for securing to designated points on the primary sequence of the target protein. By stitching the chain together in this manner, the protein is protected from denaturation and from wiggling away from its desired conformation.

Thus, in one aspect, the invention is directed to a method to control conformation of a peptide in solu¬ tion, which method comprises: providing a pair of linking groups at desired points in the sequence of the peptide, and conjugating said linking groups to each end of a rigid molecule or each end of a rigid portion thereof, containing, at said either end, compatible linking groups. In another aspect, the invention is directed to molecular sticks which are either themselves rigid molecules containing linking groups at either end, or molecules having rigid portions containing, at either end of a rigid region, such compatible linking groups.

In still another aspect, the invention is directed to proteins or peptides having a conformation which is secured by the foregoing molecular sticks. In still another aspect, the invention is directed to peptides containing, at designated locations along the primary sequence, at least two specific linking groups suitable for attachment to the molecular stick. Brief Description of the Drawings

Figure 1 shows a conceptual view of the molecular stick and its use to secure a desired three- dimensional conformation.

Figure 2 is a graph of I- values for a target protein derivatized with a series of molecular sticks of varying lengths.

Modes of Carrying Out the Invention

A. Definitions

The ''molecular stick" of the invention is defined as a molecular entity having a rigid "spacer" por¬ tion which has at either end thereof a specific linking group suitable for attachment to appropriate points along the sequence of the primary protein structure. The spacer portion, which must be rigid, may be the entire molecule used as a securing bar for the three-dimensional conforma¬ tion, or may be a portion of a larger molecule. By stat- ing that the functional linking groups are at either end of the spacer portion of the molecular stick, it is intended that the spacing between these groups be such as to result in the desired conformation when bound to the primary structure. The rigid portion of the molecule may extend farther, however, at one or both ends past the "spacer" portion; outside regions of this molecule are basically irrelevant to the function of the stick. Thus, the molecular stick contains a spacer which is framed by functional groups, but the molecule in which the spacer occurs may be extended at either or both ends by a further rigid portion, or by a flexible extension, according to preference.

Thus, as used herein, "molecular stick" refers to a molecule which comprises a rigid structure of a length as required to join the desired positions on the target peptide or protein framed at either end by a link¬ ing group capable of conjugation to a complementary link¬ ing group on the desired peptide or protein.

The spacer portion of the stick is characterized by having a rigidity of order n _> 10, where n is the denominator in the

360 descriptor which represents the number of degrees the n stick may bend from a straight line or other specified shape. Thus, the larger the value of n, the more rigid the stick.

"Conformation status" refers to the three- dimensional shape of the molecule in question. As most peptides and proteins are single-chain amino acid polymers, this chain can be bent, twisted, and rolled up into a variety of three-dimensional configurations. The "conformational status" refers to the three-dimensional shape which results when a particular conformation of this chain is assumed. "Designated points on a target protein" refers to ^"positions arbitrarily chosen in order to effect the desired conformation along the primary structure.

"Target protein" refers to a peptide or protein whose primary sequence is or is to be folded into a desired three-dimensional conformation.

"Compatible" linking groups refers to those which are capable of achieving ligation between the substances to which they are attached. The linking groups may be directly reactive with each other, such as, for example, an amino and a carboxyl group which can be linked to form an amide, or two sulfhydryl groups which can be linked to form a disulfide. In the alternative, these linking groups are still considered compatible if they can be ligated by means of a suitable linker per se. For example, the linking groups at designated points on the target protein may be amino groups, whereas the linking groups on the molecular stick are sulfhydryls; these can be made compatib^τe through use of a heterobifunctional linker such as S___.-_.CC. Or_Λ the linking groups at the designated points on the target protein might be amino groups to be made compatible with a molecular stick also containing amino groups . This could be effected by a homobifunctional linker such as glutaraldehyde.

B. General Description

It is the aim of the invention to provide a means to assure a higher preponderance or probability of particular range of three-dimensional shape based on a target protein primary structure. This result is achieved by binding together designated points along the primary sequence of a desired target protein using a "molecular stick" having a spacer of the appropriate dimensions to hold these points together in a manner which controls variability and increases the probability of a derived conformation over time.

The length of the spacer portion of the molecular stick will be determined by the target protein. The length appropriate in a particular case will, of course, depend upon the choice of the designated points in the target protein, and on the nature of the three- dimensional conformation desired. The spacer portion must be rigid along this length — i.e., it must_r itself, as¬ sume either a linear conformation which does not deviate substantially, or a particular curved conformation which is desired to effect the desired conformation in the target protein. In general, the rigidity of the spacer must be of an order greater than or equal to 10, wherein "order" is defined as above. The higher the order, the more effective the control which can be obtained by inser- tion of the molecular stick in attaching it to the designated .points on the target protein.^' Thus, while molecular sticks containing spacers characterized by orders 10 or greater are sufficient to fall within the scope of the invention, those of higher orders, for example, 15 or 20 or even 25, are preferred.

It should be recognized, however, that despite the rigidity of the spacer portion of the stick, the target peptide or protein will not, itself, be correspond¬ ingly rigid. Indeed, generally speaking it is not neces- sary, and perhaps not desirable, to achieve a completely rigid conformation of the target protein. The desired goal is to reduce the number of conformational states the protein can assume so that the statistical probability of a particular molecule assuming the desired conformation at a particular time, or assuming a conformation within ac¬ ceptable limits of that desired at a particular time, will be acceptably high.

In general, the compositional choices for the molecular sticks are very large, but a typical generic formulation comprises a polymer composed of the spacer region bounded by two linker regions. The spacer portion can conveniently be constructed of repeating monomer units of identical nature, and the length of the spacer can thus be adjusted by the number of monomer units in the polymer. The linking units placed at either end of the spacer will contain convenient functional groups for attachment to, and therefore compatible with, the functional groups oc¬ cupying the designated points in the target protein.

There are a variety of choices for the spacer portion of the molecular sticks. First, certain proteins are themselves stable as alpha-helices or beta-pleated sheets. The alpha-helical configuration of the protein sticks can further be stabilized using the hydrogen bond alpha-helix mimics of Arrhenius (supra) . These can be varied in length by altering the number of amino acids. Second, nucleic acid complexes of various sizes and constructions have been shown to be relatively stiff and to provide defined separations. For example, duplex DNA shorter than about 500 basepairs is just small enough to be too stiff to circularize. Thus, 500 bp has an order of 1; a 50 bp DNA segment would have the required minimum order of 10. In addition, cross-linked dextrans, contain¬ ing on the order of 25 monomers, are commercially avail¬ able, and also have the required rigidity, or can be made to have such by using unsaturated linkage between monomers. As is the case with respect to DNA, the rigid¬ ity and length of the dextran spacer portion can be varied by altering the number of glucose units in the polymer.

In addition to polymers of biological origin, it may be convenient to use molecular sticks comprises of monomers of nonbiological origin, such as polybutadiene, polyethylene oxide, and the like. This is convenient, especially if the molecular stick is sufficiently short that oligomers of these residues provide sufficient rigid- ity. Longer nonbiological spacers can be synthesized by using conformationally restricted monomer units such as ring compounds.

A table of molecules having well-defined distances between specific points on their surfaces is set forth in Stryer, L., Ann Rev Biochem (1978) 47:819-846, incorporated herein by reference. This list includes various transfer RNAs, such as yeast phenylalanine tRNA, E_- coli f-met tRNA, and E _ coli glutamate tRNA, as well as ribosomes and a variety of other proteins. Proteins of these characteristics include, for example, various oligomers of 2-hydroxyethyl- -glutamine_Λ transferrin, carboxypeptidase A, thermolysin, rhodopsin, galactose receptor, immunoglobulin G, calcium-bound ATPase, gramacidin A, RNA polymerase, pyruvate dehydrogenase complex, cytochrome oxidase, chloroplast coupling factor, bacterial luciferase, aspartic transcarbamylase, and myosin. Also included are microtubules, various protease inhibitors, erythrocyte membranes, and aspartokinase. By a suitable choice among the foregoing and other rigid molecules or rigid portions thereof, molecular sticks of the desired length and rigidity can readily be obtained.

As set forth above, the molecular stick may constitute substantially the entire molecule used to link the designated points on the specific protein targeted for three-dimensional control, or the rigid spacer can constitute only a portion of this molecule. The rigid spacer portion which is included between the functional groups which will be used to link the stick to the primary protein structure is the length-determining portion of the molecular stick. The spacer may therefore be only a small portion of the entire molecule. For example, long-chain dextran or nucleic acid molecules can be used, but the linking functional groups placed within the specified distance of each other, leaving the remainder of the molecule outside the spacer frame. The remaining portions of the molecule, as stated above, can either be rigid or flexible, or may contain regions of each characteristic. The provision of the linking functional groups is arranged by providing compatible groups on either end of the spacer which are complementary to groups residing in or attached to the target protein at the designated locations. Clearly, a wide variety of choices is avail¬ able. In one easily envisioned example, a cysteine residue may be placed in the primary protein sequence at the desired points along the chain in order to provide sulfhydryl groups for binding. This method is best utilized when the target protein does not itself contain sulfhydryl groups necessary to stabilize its native conformation. However, even in this case, by proper design of synthesis, sulfhydryl groups might still be employed in forming the linkage.

Suitable groups for linkage also include C-terminal carboxyl groups and N-terminal amino groups, since side-chain carboxyl and amino groups can be retained in their protected form after protein synthesis by utilization of the method of Nakagawa, S.H., et al, J Am Che Soc (1985) 107:7087-7092. By utilizing this method, only the terminal amino and carboxyl groups will be avail¬ able for binding to the linking agent or to the ends of the molecular stick.

The molecular stick, of course, will also be provided with groups capable of forming linkages at either end of the spacer. Such groups also can be chosen from amino, carboxyl, aldehyde, sulfhydryl, and the like.

Depending on the protocol, the molecular stick and target protein may be linked directly using the functional groups provided, or can, more usually, be linked using a commercially available or other type of linker, including heterobifunctional linkers. Such linkers are available, for example, from Pierce Chemical Company. Linkers include N-succinimidyl-3-(2-pyridyl thio)propionate (SPDP), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), and sulfonated or unsulfonated maleimidobenzoyl succinimide ester (MBS) . All of the foregoing provide a labile ester for reaction with amines to form amides and a double bond for reaction with sulf ydryl. A variety of intermediate functional groups which can be specifically attached to particular amino acid residues in proteins is given by Feeney, R.E., et al, in "Modification of Proteins", R.E. Feeney, ed. , Advances in Chemistry Series, 198 (1982), M. Juan Comstock, ed., American Chemical Society, Washington, D.C., pp. 3-55, incorporated herein by reference. In Table 1 of that article, on pages 10-11, are given specific reactivities of particular amino acid side chains with a variety of reagents.

Derivatization of dextrose or dextran or other polysaccharides for binding to other moieties is described by May, F.M., et al, in "Separation and Purification", 3d ed., Perry, ed., in "Techniques of Chemistry" (1978), l_2:257-293, J. Wiley, New York.

A particularly useful linkage strategy for double-stranded DNA spacers employs derivatized bases, in particular, derivatized thymidine. Various techniques for derivatization of nucleotides are known in the art. For example, Langer, R., et al, Proc Natl Acad Sci USA (1981) 7_8: 6633-6637 , described thymine or uracil residues having the 5 '-position derivatized to an allylamine linker arm. The amine can then be reacted with a subsequent linker, if desired, such as MBS, to provide a functional group compatible with a sulfhydryl. Nucleotides derivatized to mercury have also been described (Dale, R.M.K., et al, Biochemistry (1975) L4:2447-2457 ; ibid: 2458-2469) .

In preparing the molecular sticks, a single- stranded DNA is provided hybridized to a primer and then extended using a nucleotide mixture containing the derivatized moiety. Bases which are complementary to the derivatized nucleotide are spaced in the single-stranded extension to provide the molecular stick of the desired length. For example, if derivatized thymidine residues are employed, the single-stranded extension will contain a pair of adenine residues spaced at the desired distances; the remainder of the residues in the single-stranded extension will be non-adenine residues--i .e. , guanine, cytosine, or thymine.

The nature of the linking groups is not significant, so long as the groups are compatible with each other in the same molecule, and complementary to each other with respect to the molecular stick and the target protein. As is evidenced above, a wide variety of choice can be made.

In carrying out the method of the invention, the points designated on the target protein which ought to be linked in order to secure the proper conformation, and the distance required between these linked residues is determined, in advance, if possible, but more usually by systematic variation of various designated point locations on the target and stick spacer length, followed by assay of the resulting conformation.

For predetermination, this is accomplished, for example, by examination of an X-ray crystallographic representation of the target protein, thus determining the appropriate spacing between residues in the undenatured form. This approach is, of course, available only if, indeed, the X-ray crystallographic study of the protein has already been done.

In the alternative, the appropriate stick spacer length and location of target points can be screened for using a simple assay procedure. Molecular sticks in a range of several spacer lengths are used to obtain controlled conformations of a target protein. (The designated linking points of the target protein can also be systematically varied. ) The test protein set is then subjected to a suitable bioassay to determine reactivity; for example, immunoreactivity with the appropriate immunoglobulin or ability to bind receptor may be the required property. Standard ELISA or radioimmunoassay procedures can be used to determine the degree of binding in these assays. If the protein is an enzyme, enzyme activity can be used as a measure. Proteins having selected points bound by sticks which vary in length by factors of, for example, five or ten, leads to a modest number of representative examples which permits a focusing on the appropriate length for the stick. The required bioassays and immunoassays are well known in the art.

Statement of Utility The proteins prepared by the method of the invention are useful in a number of applications, includ¬ ing reagents for diagnostic immunoassays; immunogens for elicitation of antibodies, and therefore in vaccines; in therapeutics as drugs which are capable of binding to desired receptors, and so forth. In any situation in which control over the three-dimensional conformation of the protein is required, the methods and compositions of the invention can be used to reduce excess conformational freedom by constraining the structure to the desired three-dimensional structure.

In this connection, it is to be noted that protein mimics can be found for a number of functional compounds which may themselves be other proteins, or, more interestingly, may be totally nonprotein in nature. Two well-known examples are the sweetener aspartame, a dipeptide which mimics the ability of sugar to bind to a receptor which registers sweetness, and the dipeptide L- ornithyl taurine, which is capable of binding to salt receptors (Tada, M. , et al, J Aqric Food Che (1984) 3_2 : 992-996) . It is also well known that the short enkephalin peptides can mimic opiates in relieving pain. The molecular sticks and methods of the invention are particularly useful in providing the appropriate conforma¬ tion profiles to peptide mimotopes which are designed to mimic various drugs and vaccines. The primary structures of these mimotopes can be determined and prepared as described, for example, by Geysen, H.M., et al, in PCT ap¬ plications WO86/06487 and WO86/00991.

It has also been shown in copending application Docket No. 2550-0004, incorporated herein by reference, that the paratopes of immunoglobulins can be mimicked by synthetic "paralogs". These paralogs can also be fine- tuned to higher specificity using the molecular sticks herein.

Example 1 Synthesis of Molecular Sticks Molecular sticks of varying lengths are synthesized using a series of mostly G-C double-stranded DNA polymers with varying spacing between thymine residues derivatized with linking arms suitable for protein attach¬ ment. The synthesis is as follows:

The following series of primed DNA sequences is prepared using standard solid-phase DNA synthesis techniques for the single-strand followed by annealing to primer. This portion of the molecules contains ordinary nucleotide units. For convenience in analysis, Aval and Hpall restriction sites are included as shown on the single-stranded portions of the starting primed sequences. Aval Hpall

5'-CTCGGGTACCGGACCTGGTG-3'

GGACCAC

5 ' -CTCGGGTA-G-CCGGACCTGGTG- 3 '

GGACCAC

5 ' -CTCGGGTA-GG-CCGGACCTGGTG-3 '

GGACCAC

5 ' -CTCGGGTA-GGG-CCGGACCTGGTG-3

GGACCAC

5 ' -CTCGGGTA-GGGG-CCGGACCTGGTG-3 '

GGACCAC

5 ' -CTCGGGTA-GGGGG-CCGGACCTGGTG-3 '

GGACCAC

5 ^r -CTCGGGTA-GGGGGG-CCGGACCTGGTG-3 '

GGACCAC The DNA sequences shown above will generate double-stranded sequences having tether points at varying spacings when the primer is extended using a nucleotide mixture which includes thymine derivatized so as to provide linking arm. The complementary thymines to the two unpaired adenine residues shown in the list will thus be spaced from 4 to 10 nucleotides apart, depending on the number of intervening guanine residues appearing in the primed single-strand at the upstream end of the Hpall site.

Each of the listed primed DNAs is extended by treatment with E. coli polymerase I using a mixture of nucleotides in which the thymidine triphosphate is derivatized to sulfo-M-maleimidobenzoyl sulfo-succinimide ester (sulfo-MBS). The MBS-derivatized thymidine is prepared by reacting a dUTP modified at C5 with a C7-NH.- spacer (Clontech, Palo Alto, CA) with sulfo-MBS, available from Pierce Chemical Company (Rockford, IL) . The MBS- derivatized thymidine is then capable of further reaction with sulfhydryl groups to provide thioether linkages to a target peptide.

The synthesis is initially conducted using labeled nucleotides and confirmed by cleavage with Aval and Hpall. The synthesis is then repeated using unlabeled nucleotides to obtain the series of molecular sticks. As the nucleotide residues have a spacing of approximately 3.4 A°/residue, the foregoing series contains spacings of 13.6-34 A , approximately.

Example 2

Stabilization of Peptide Conformation

A. The peptide sequence: Cys-Ala-Asp-Pro-Tyr- Glu-Glu-Gly-Asp-Asp-Gly-Arg-Thr-Cys is synthesized using standard solid-phase techniques. Monoclonal antibodies reactive with this peptide are prepared by conjugation of the peptide to carrier, injection into mice, and immortalization of spleen cells followed by screening with the peptide. A suitable monoclonal antibody or set of monoclonal antibodies is chosen for use in evaluating the efficacy of the molecular stick in maintaining the peptide conformation. In the alternative, polyclonal antisera could also be sued for this evaluation.

Samples of the peptide are reacted with each of the molecular sticks using the method described by Liu, F.T., et al, Biochemistry (1979) 18^690-697. Ap¬ proximately 1 mg of each derivatized peptide is prepared. Synthesis of the desired tethered peptides can be confirmed by subjecting a sample of each preparation to nonreducing polyacrylamide gel electrophoresis with and without cleavage with Hpall and appropriate development of the gels .

Each of the restricted peptides is then tested for affinity to the antibody preparation using a series of dilutions in standard ELISA techniques. In an illustra¬ tive protocol, microtiter wells are coated with the anti¬ body preparation, and the inhibition of binding of the target peptide labeled with I 125 is measured at varying concentrations of the tethered peptides. A value for I_5Q (the concentration of the tethered peptide at which the binding of the labeled peptide is inhibited by 50%) is calculated for each tethered peptide.

In an alternative assay, the antibody prepara- tion may be labeled with I 125 and the free target peptide attached to Immunodyne. The tethered peptide is then added in varying concentrations to target peptide spots . The labeled antibody is then added to determine its abil¬ ity to bind the bound target peptide in the presence of tethered protein. The I,-_n values are then plotted as a function of length of molecular stick. A curve similar to that shown in Figure 2 is obtained. As shown, very long sticks have I - values approximately equivalent to that of the free peptide since they are too long and rigid to bind to both designated points on the target protein. Since only one designated point is bound, the conformation of the bound protein is substantially the same as they of the underivatized protein. However, a minimum value is obtained for the stick of correct length to restrict the protein to the correct conformation status when bound to both designated points.

B. In a similar manner, molecular sticks of the correct length are determined for the peptide sequences: Pro-Tyr-Cys-Asn-Tyr-Ser-Lys-Tyr-Trp-Tyr-Leu-Glu-His-Ala- Lys-Cys and Cys-Ala-Gly-Ser-Ala-Gly-Gly-Ser-Cys-Val-Pro- Gly.

Claims

1. A method to control conformation of a target peptide in solution, which method comprises: providing a pair of first linking groups at designated points in the sequence of said target peptide; and conjugating said linking groups to a molecular stick containing, at each end of the spacer portion thereof, a pair of second linking groups compatible with the first linking groups.

2. The method of claim 1 wherein the spacer portion of the molecular stick is an oligomer of monomeric units.

3. The method of claim 2 wherein the monomer units are amino acids or nucleotides.

4. The method of claim 1 wherein the spacer portion of the molecular stick has a rigidity order of at least 10.

5 The method of claim 1 wherein the second linking groups are 'amino groups.

6. The method of claim 1 wherein the second linking groups are capable of binding sulfhydryl groups.

7. The method of claim 6 wherein the second linking groups are provided by a heterobifunctional linker which contains a hydroxysuccinimidyl ester for binding to amino groups and a maleimide residue for binding to sulfhydryl groups.

8. The method of claim 1 wherein the target protein is .selected from the group consisting of a mimotope for a drug and a mimotope for a vaccine.

9. The method of claim 6 wherein the first linking groups are sulfhydryl groups.

10. A molecular stick useful for stabilizing conformation of a protein molecule which comprises a rigid molecular spacer portion framed by linking groups.

11. The molecular stick of claim 10 wherein the spacer portion of the molecular stick is an oligomer of monomeric units .

12. The molecular stick of claim 11 wherein the monomer units are amino acids or nucleotides.

13. The molecular stick of claim 10 wherein the spacer protein has a rigidity order of at least 10.

14. The molecular stick of claim 10 wherein the linking groups are amino groups or derivatized amino groups .

15. A composition of matter which comprises a conformationally stabilized target protein consisting es¬ sentially of a target protein and a molecular stick, wherein designated points on the primary structure of said protein are linked by means of the molecular stick.

16. The composition of claim 15 wherein the spacer portion of the molecular stick is an oligomer of monomeric units .

17. The composition of claim 16 wherein the monomer units are amino acids or nucleotides .

18. The composition of claim 15 wherein the spacer portion of the molecular stick has a rigidity order of at least 10.

19. A target protein which comprises a primary sequence of amino acids containing, at two designated points therein, a pair of first linking groups capable of binding to a pair of compatible second linking groups at each end of the spacer portion of a molecular stick.

20. The composition of claim 16 wherein the first linking groups are sulfhydryl.