University New Haven, Connecticut Yale
I
- - -- - - - -h e c u l a t i o n s based o n molecular models
Although accurate molecular models were very helpful in developing the present concepts of protein and nucleic acid secondary and tertiary structure, the study of peptide conformation with molecular models has been classified as naive ( 1 ) in comparison t o the experimental methods of physical chemistry and crystallography. Presumably the computer approach ( 2 ) lies between these "extremes," allowing a very rapid search among the almost infinite number of possible conformational isomers for the most favorable steric relationships. These studies have indicated that steric hindrance limits peptide conformation to a much greater degree than was previously assumed. I n certain respects, space filling molecular models function like simple computers programmed lo climinate unfavorable conformations. This is because average bond lengths and angles are "built-in" and steric requirements are served in prouortion t o the size and hardness of component parts and limits of connector elasticity. Within these limitntions, all conformations allowed by accurate madels are relatively t h e r m o d y n n m i c n l l y Figure 1. Components for orrembling of po1ypep)ides. Left to right, mod& top row: Hydrogen, methyne, methylene (or ornino), methyl (or ammonium); second row: peptide bond, divolent oxygen, divalent sulfur, carbonyl; lhird row; phenyl, imidorole, indole; bottom row; guonido, prolyl (for 011 but N-terminal poritionr).
favored. The difficulty, of course, lies in finding the most favored conformation-if indeed only one exists even for a set of closely defined conditions. Such a conformation should best represent the three dimensional st,ructure of the molecule in question. Here, important advantages of the comput,er approach become apparent: virtual models can be assembled, analyzed, and disassembled rapidly and effects of small variations in bond lengths and angles and Van der Waals' radii quickly determined. However, real models are st,ill useful-for those without the means for computer studies, for developing, testing, or display of concepts of conformation and molecular interaction and for the interpretation of X-ray diffraction data. The space filling models described in t,his paper (3) were designed specifically for the construction of polypeptides. ;\lolecules compriscd of the familiar amino acid residues can be built from various combinations of the 13 componc~$s shown in Figure 1. The scale is small, 0.5 em = 1 A, and the uuits, composed of a white polystyrene-rubber plastic, are corinected by integral stainless steel pins or sockets. Molded clusters of atoms are used where possible, sharply diminishing the drudgery and expense of handling large numbers of atoms in the assembly of macromolecules. Bond lengths and angles represent averages of values culled from the literature (Fig, 2). Peptide bond dimensions are those of Pauling and Corey (4) wit,h Van der Waals' radii of carbonyl oxygens and imino hydrogens shortened along the axis of potential hydrogen-bond formation t o allow an internuclear C. . .N distance of 2.8
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,amino
, A,-
terminus. 0 chain
0, disulfide
-AG-Alldisulfide (hidden)
-amino terminus, A - chain -1ysine 29 of 0-chain
Figure 4. th; left.
Model of a
backbone chain conformation of
bovine insulin. Left and right: front and
was not considered although it does not appear excludable on stereochemical grounds. Although there are no experimental data pertinent t o the structures proposed for gramicidin-S, another cyclic peptide, vasopressin, has been tested for the presence of hydrogen bonds. This peptide hormone contains a 21-membered ring closed by a disulfide bridge; a model containing a pair of transannular hydrogen bonds is shown in Figure 3, bottom. A kinetic study of hydmgen-tritium exchange in lysine vasopressin was used to test this hypothetical structure (17) and showed that all hydrogens exchange rapidly, indicat,ing that hydrogen bonds are riot present. Such data are not unequivocal, however, because exchange processes are acid and base catalyzed, and lysine vasopressin contains a basic lysyl residue a t position 8 in the chain. This effect could obscure t,he presence of hydrogen bonds. A model for vasopressin was proposed very recently by Gibson arid Scheraga (IS) based on computer evaluat,ions of energy parameters. Their structure did not include hydrogen bonds, although the confnrmat,ion illust,rated in Figure 3, bottom, cannot be excluded on purely stereochemical grounds. It should be st,ressed that the intention here is not to offer final argument,^ for or against hydrogen bonds in cyclic peptides or to evaluate structures proposed by ot,her workers but to demonstrat,cthe best use of models which is to raise que~t~ions or propose hypotheses that can be t,ested or answered experiment,ally. The models have also been employed to consider the structure of a small protein, insulin. Although this hormone was crystallized forty years ago and its primary structure has been known for twelve years, very little is linowri about. the secondary and tertiary struct,ure of this molecule. A structural model was presented more than a decade ago (19) and a new model is presented here which incorporates more recent experimental observations: i.e., insulin dimers are formed by intermolecular disulfde exchange between two intra-chain disulfide loops (20); zinc-insulin cou-
rear
views of t h e model;
center:
diogrom of view
shown on
sists of twozinc atoms bound to t,hrce insulin dimers (21) ; the site of zinc binding is either the amino-terminus of the B-chain (22); or one of the histidine residues (23); the amino terminus of the A-chain is close t,o lysine-29 of chain B ( 2 4 , helical content appears lo be of the order or* 2445% based on circular dichroism (26) or optical rotatory dispersion (26) measurements, rcspectively. The backbone chain of a niodel co~isistentwith the above data, and also most of the older literature, is shown in Figure 4. The B-chain is in tho form of a right handed a-helix from cysteine-7 to proline-28 in agreement with Yang and Doty (26), although a shorter helix could be formed by unwinding one or both ends of the B-chain. B-chain residues included within the interchain disulfide loop in helical array represent about 25y0 of the molecule. Unwinding the chain outside of the disulfide loop would not disturb the overall conformation of the molecule. Figure 4 shows the A-chain not parallel to, but twisted about the axis of the B-chain. The small intrachain disulfde loop is thus exposed for intermolecular disulfide exchange. I n the model, the same loop shields histidine (B-10) suggesting that the imidazole moiety involved in zinc binding is probably that of histidine B-5. Alternatively, the lack of susceptibility of zinc insulin to leucine aminopeptidase (27) would suggest t,hat amino t,erminal phenylalanine (B-1) is the ligand involved. If the zinc-insulin hexamer is closely packed with the folded A-chain portions in the center of the complex, susceptibility to leucine aminopeptidase might be completely blocked because of steric hindrance. Figure 5 shows views of the insulin model with all side chains attached. Whether such a model is a t all related to the true conformation of insulin remains to be seen. I t has been demonstrated, however, that a model of insulin incorporating much of the experimental data available in the literature can be constructed-even under thc Volume 45, Number 9, September 1968
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589
",.,..E."
I
0.0
I
I.,.
I
Figure 2. Linear ond angular dimensions of atoms in the components illurtroted in Figure 1.
0
A in -C-0. . . H-N-. For the sake of convenience in handling the models, carbonyl oxygens were grooved to allow a limit,ed rotation of groups adjacent to this moiety. Similarly, edges of sulfur atoms were grooved so that rotation of RS about the S-S axis of RS-SR could occur. Because the connectors are non-deformable and faces of bonded atoms touch, the models are less flexible and exhibit more accurately the effect of nonbonded interactions in limiting peptide conformation. I n this respect the models mirror conformational limitations described in steric maps (2). One can observe how interactions of adjacent peptide bonds allow only one half the possible backbone chain conformations in dipeptides and how side chain-backbone chain interactions restrict the number of conformations even more (5). Because faces of bonded atoms touch, only certain positions of y atoms are possible relative to CmCg bonds or to the backbone chain as was described by Ramachandran, et al. (6). These properties impart to the models a diminished flexibility aud limit the model to conformations most sterically favored and most representative of the contemporary view of polypeptide structure. It is on this basis that the following speculations on structure are offered. Although considerable physical and chemical data suggest that linear peptides in solution are not random 7 - 1 1 ) prediction of probable peptide conformation from primary structure is presently limited to cyclic structures. This is because the number of allowable conformations of linear peptides is still great in spite of extensive restrictions imposed by backbone-backbone and backbone-side chain interactions. Examples follow in which the models are used to analyze possible conformatious of cyclic peptides. Hydrogen bonds are important in stabilizing the secondary structure of biopolymers. Therefore, the effect of hydrogen bonds on conformation-and possibly'activity of peptides-is of vital interest. Although results vary with technique and amino acid composition, experiments with linear peptides (12) have indicated that more than twelve residues must be present before hydrogen-bonded structures are found in significant amounts in aqueous solution. Because of inherent conformational limitations of the entire molecule, similar bonds in cyclic peptides might be more stable and thus more important in determining conformation. I n Figure 3, center, is shown cyclohexaglycine containing a pair of transannular hydrogen bonds. Since a model taking this form can be assembled, there are apparently no important steric restric588
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Jourwl o f Chemicol Education
Figure 3. Models of cyclic peptide.. Upper, gromicidin-S; center, ~ycloheroglycine; bottom, lyrine v ~ r o p r e ~ ~ i nOnly . backbone chains are depicted, except for glycine.
tions to the conformation illustrated. Perhaps relevant to the question of stability of such bonds in this case are X-ray studies of cyclohexaglyciue ( 1 3 ) . The conformational isomer shown in Figure 3, center, is indeed fou~ld-but in the same crystal other forms are also observed containing peptide bonds hydrogenbonded to water rather than to each other. This indicates that intramolecular and intermolecular hydrogen-bonded structures do not differ significantly in stability even in crystalline form. I n predicting stable structures by computing conformational energies, the effect of a hydrogen bond on calculated energy levels %ill be more significant in small molecules than in large ones. Thus, in the mathematical determination of stable conformers of small peptides, the relat,ive importance of hydrogen bonding mag be given more weight than is actually warranted. This might explain why computer based predictions of the conformation of gmmicidin-S, reported independently by two groups (14, 15), had little in common except for the presence of two hydrogen bonds. Illustrations of the structures proposed for gramicidind, a cyclic decapeptide, can be found in the references cited; Scheraga's group suggested, for gmmicidin-S, a b-pleated sheet structure containing a pair of transannular hydrogen bonds, while Liquori's group favored one containing a pair of single turns of an ahelix in antiparallel array. In studying these proposals with the aid of the models described in this paper, it was possible to build the pleated sheet described in (14) but not the other, although both could be huilt with Corey-Pauling-Koltun (16') models. In addition, it was possible to build a model of gramicidin-S that contained three rather than two transannular hydrogen bonds (Fig. 3, top). In (14) and (15) such a structure
Figure 5.
M o d e l of bovine insulin with all ride choinr ottoched.
Views ore similar to those in Figure 4.
conditions of extensive conformational restrictions imposed by the design of the model introduced in this paper. Furthermore, as compared to similar structures built from other commercially available space-filling models, the present model is much smaller, without loss of detail or accuracy, requires manyfold fewer parts, and can be assembled a t about one fifth the cost.
(8) IT.\RDY, P . >I., KENNEII,G. W.,
A N D SHEI~I'.\RD, 1:. C., Tetrahedron, 19, 95 (1063). ( 9 ) BOVEY,F. A,, AND TIERS, G. V. D., J . A m . C h m . Soc., 81, 2870 (1959). ( 1 0 ) WIELIND,T., A N D BENDE,H., Chem. Ber., 98, 504 (196,5). It., N I ) SIEIIEIL, P., Helo. Chim. Acla 40, 624 ( 1 1 ) SCHIYYZEB,
,.,>.,,,. 110i7,
( 1 2 ) FASMAN, (;. I)., Biological fila~r0mo1e~i~le.v, 1 (Polyamitic acids), 490 (1967). J., A d a C T ~ S I 16, . , OG9 (1963). (13) KAIILE,I. L., .AND KARLE, Acknowledgment , IJEACII,S. J., NEMETIIY, C:., SCOTT,It. A., (14) \ ' . \ ~ l ) ~ : l t r < ~ O lG., A N D SCHEIL.\G.\, IT. A,, Bioehemistrg, 5, 2991 (196G). The author is a recipient of Career Development ( 1 5 ) I,~nunnr,A. XI., DE S.\NWS,P., KOY.\CS,A. I,., A N D M.\zzAAward 1-K3 An" 25757-02, United States Public Health ILELLA, L., I\'atlire, 211, 1039 (1066). W. L.,. Biopo1umei.s. (16) KOLTI~N, Service. . . . 3,. 665 (196.5). (17) EMERY,T., Personal commnnicat,ion. (18) G I ~ S O NK. , I)., AND SCHERAOA, 15. A,, PTOC.Nail. Acad. Literature Cited Sei., 5,1317 (1967). H., AND ROLLETT,J . S., Bioehim. Biophys. A d a , (19) LINDLEY, ( 1 ) SCHELLMAN, J . A,, AND SCHELLMAN, C., in "The Proteins,'' 18, 183 (1955). (2nd Ed.) (Editor: NEURATH,H.), Academic Press, (20) MARKER,K . , AND GRAAE,J., A d a C h m . Seand., 15, 565 Ine., 1964. (1961 R AN., N ,R.\MAKRISHNAN, C., A N D S A S ~ ~ E K ( 2 ) R ~ M ~ C H ~ N DG. ~- ~\. - , ~ ( 2 1 ) SCHLICHTKRULL, J., Acta C h m . Seand., 10, 1455 (1956); HARAN, Y., in aspect^ of Protein Sirueture," (Editor: CUNNINGHAM, L. W., FISCHER, 11. L., A N D YESTLING, ~ A M A ~ H ~ N D R A G. N , N.), Academic Press, Ine., New C. S., J . A m . Chem. Soc., 77, ,5703 (3955). York, 1963, p. 121. R , Acta Chem. Seand., 14,2071 (1960). (22) M ~ R K E K., (3) The models described in this paper arc available from C., AND EPSTEIN,J., J . A m . Chem. Soe., 76, 2170 (23) TANFORD, Edmund Scientific Company, lrll E. (:lmrester Pike, Barrington, New Jwsey. (1954); BRILL,A. A,, AND VENABLEJR.,J. H., J. A m . Chem. Soe., (19671, in press. ( 4 ) PATILING, L., A N D COREY,R . B., Pvoc. Roy. Soc., B 141, In. c~i s-h.-,. ( 2 4 ) ZAKN,H., 6th Intern. Congr. Biachem. New York, August, --, -: n 1964. -~ ( 5 ) LEACH,S. J., NEMETHY, G., ANI) SCIIERAG,~, IT. A,, nio( 2 5 ) MEKCOLA, I). A,, MORRIS,J . W. S., ARQUILLA, E. R., AND polvmers, 4 , 369 (1966). BHOMEH,W. W., Bioehim. Biophys. A d a , 133, 224 (6) MACH AND RAN, (;. N., A N D J ~ , k c ~ s H ~ M i N . i n . \ uA. N i\'~., , (1967). Biopolymers, 4 , 495 (1966). J. Am.Chem. Soe., 79,761 (1957). ( 2 6 ) YANG,J.T., ANDI>OTY;P., E., J . A m . Chem. Soc., 78, 369 (19%); ( 7 ) EILENIIOGEN, LT, N. C., ~ ~ I L L E RG. , W., SOLONY, N., A N D
