Proc. Nati. Acad. Sci. USA Vol. 87, pp. 871-875, February 1990 Biophysics Design of crystalline helices of short oligopeptides as a possible model for nucleation of a-helix: Role of water molecules in stabilizing helices (x-ray diffraction/amphipathic hefices/protein folding) R. PARTHASARATHY*, SANJEEV CHATURVEDI, AND KUANTEE Go Center for Crystallographic Research and Department of Biophysics, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263 Communicated by Herbert A. Hauptman, October 30, 1989 (receivedfor review August 7, 1989)

ABSTRACT We have designed, synthesized, crystallized, oligopeptides isolated from helix-containing regions of pro- and performed x-ray analysis of several hydrophobic tripep- teins gave negative results (8, 9). tides that show an extended near a-helical structure in the It has been rather widely assumed that short oligopeptides crystalline state. All of the tripeptides that show this remark- will not have a preferred and well-defined conformation. For ably stable helix crystallize with two or three water molecules; example, it has been shown that a given sequence in a peptide they all have glycine at the N terminus and have increasing does not always assume the same conformation in different hydrophobicity as one moves from the N to C terminus. Even proteins (10). Also, the Zimm-Bragg equation (11) predicts though three residues in the oligomer are not sufficient to that peptides as short as 20 residues should not show mea- complete a turn, one of the water molecules acts as an added surable a-helix formation in water regardless of the temper- residue and links up adjacent tripeptide segments along the ature and amino acid sequence (12). However, Lerner and his helix axis so that in the crystal, the helix appears effectively as coworkers (13) have found that a highly immunogenic non- one long continuous helix. Two of these tripeptides are stabi- apeptide from influenza virus shows structure that is mea- lized by two water molecules that enable the peptides to surable by NMR. Further, a-helices of short peptides of complete a turn of the helix and extend the helical structure particular sequences have been found to be stable and their throughout the crystal by linking translationally related pep- stability in the solution state has been attributed to be due to tides by hydrogen bonds. In two other peptides, these roles are salt bridges (14, 15); a similar conclusion has been drawn for played by three rather than two water molecules. Though these the stability of a-helices in the crystalline state (16). Though tripeptides have different crystal symmetry, they all show the many empirical correlations have been put forward for amino basic pattern of hydrated helix and packing, indicating the acid preferences for specific locations at the ends of helices strong conformational preference for a stable structure even (17-19) and have been used in designing a-helical peptides for these tripeptides. Such conformationally stable hydrated (20, 21), ". .. there are few examples of short, noncyclic structures for short specific related sequences illustrate their peptides that adopt stable'conformations in solution unless possible importance in nucleating protein folding and in the they are bound to a biological receptor" (3). role water molecules play in such events. Based on our earlier work of two short peptides that are crystalline helices (22), we have now designed, synthesized, How proteins start to fold and what clues the primary and crystallized (at pH 7.0) three more tripeptides and carried structure itself gives to the folding process have long been a out x-ray diffraction studies of two of these that show stable mystery. Though it was demonstrated as early as 1960 by helical conformation in the crystalline state. Taken together, Anfinsen, White, and Sela (for a review, see ref. 1) that we now have four clear examples of tripeptides-namely, polypeptide chains can refold in vitro, the mechanism of the Gly-L-Ala-L-Phe (GAF) (22), Gly-Gly-L-Val (GGV) (22, 23), folding process and how different amino acid sequences Gly-L-Ala-L-Val (GAV) (Fig. 1), and Gly-L-Ala-L-Leu (GAL) direct chains into unique conformations have not been elu- (Fig. 2)-that are helical in the crystalline state and a fifth cidated. It is being increasingly realized (for recent short one, Gly-L-Ala-L-Ile (GAI) (whose x-ray structure determi- reviews on protein folding, see refs. 2 and 3) that not only do nation is not yet completed), that shows all of the character- the amino acid sequences determine the final folded confor- istics of the other four peptides. mation, but they also influence the folding process to reach the final folded state rapidly and with near perfect fidelity. MATERIALS AND METHODS One of the key elements of the folding process, as originally The crystal structures GAV and GAL are reported here and suggested by Anfinsen (4) and later experimentally observed those of GAF and GGV have been discussed by us earlier by Anfinsen and coworkers (5) and by many others (6, 7), is (22). GAV (C1OH19N304-3H20) and GAL (C11H21N304- the presence of nucleating sites in the polypeptide. These 3H20), Mr = 299 and 313, respectively, were synthesized by nucleating sites are short fragments ofthe polypeptide chains solution-phase peptide synthesis. Blocked tripeptides Boc- that, during the folding process, can flicker in and out of the Gly-Ala-Val-OBzl and Boc-Gly-Ala-Leu-OBzl (where Boc = conformation that they assume in the final fold; if these butoxycarbonyl and OBzl = benzyloxy) were made from the fragments have well-defined conformations, they seed effec- respective X-OBzl C-terminal residue, which was coupled tively the folding process. Anfinsen and coworkers detected with Boc-Ala and Boc-Gly by N,N'-dicyclohexylcarbodiim- the native-like folding of fragments of the polypeptide that ide/HOBt method consecutively (24). Finally, the Bzl and have highly unfavorable equilibrium constants using a con- Boc blocking groups were removed, respectively, by hydro- formation-sensitive antibody technique. Early attempts to genation and acid treatment (trifluoroacetic acid/CH2Cl2, detect stable conformations (for example, a-helices) in short 1:1). The pH of the deblocked tripeptides was adjusted to 7,

The publication costs of this article were defrayed in part by page charge Abbreviations: GAF, Gly-L-Ala-L-Phe; GGV, Gly-Gly-L-Val; GAV, payment. This article must therefore be hereby marked "advertisement" Gly-L-Ala-L-Val; GAL, Gly-L-Ala-L-Leu; GAI, Gly-L-Ala-L-Ile. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

871 Downloaded by guest on September 28, 2021 872 Biophysics: Parthasarathy et al. Proc. Natl. Acad. Sci. USA 87 (1990) a b

c

FIG. 1. GAV. (a) Stereoview of the helical turn stabilized by OWL. Note that OW1 is in the plane of the COO- group. (b) Stereoview of the crystalline helix formed by the tripeptide GAV. The side chains have been removed for clarity. The tripeptide molecules related by cell translations are linked by the water mol- ecule OW1, giving rise to the appearance of an extended helix in the crystal. OW2 and OW3 add further stability to the helix through hydrogen bonds involving N1 ... OW2 ... OW3 ... N1 of adjacent molecule. (c) Same as b but contains side chains and hydrogen atoms. and they were Iyophilized. The purity of the peptides was programs SDP'. The least-squares refinements including the checked and confirmed by running them through a reverse- hydrogen atom parameters converged to R of 0.067 (RW = phase HPLC using Delta-Pak analytical C18 column (3.9 mm 0.087) for GAV and 0.051 (Rw = 0.080) for GAL. The final x 30 cm) from Waters; a linear gradient of0-70% acetonitrile difference electron density map was featureless, with no peak in 0.01% CH3COONH4 was used for elution. Crystals of higher than 0.25 eA-3 for GAV and 0.25 eA-3 for GAL. GAV and GAL were grown by the vapor diffusion method Atomic scattering factors were obtained from the interna- from water/methanol solutions and sealed in capillary tubes tional tables for x-ray crystallography (26). Computations with mother solutions. Three-dimensional x-ray diffraction were performed on a MicroVax II. The final atomic coordi- data to the limit ofthe Cu sphere (1903 reflections, 1298 2 3oa nates have been deposited.t for GAV, and 2045 reflections, 1318 2 3o, for GAL) were measured by the w-20 scan method. The scan widths and RESULTS aperture widths were calculated using the relations (1.0 + 0.14 tanO)0 and (3.0 + 1.2 tanO)mm-1. The maximum time Conformation of the Tripeptides. All of these tripeptides, spent on any reflection was 100 s and the background count GAF, GGV, GAV, and GAL, are, in the crystal structure, time was halfthe scan time. The variation in intensity ofthree zwitterions, and each tripeptide molecule assumes a confor- standard reflections monitored every hour of x-ray exposure mation similar to a reverse turn stabilized by water mole- was <2%. The data were corrected for Lorentz and polar- cules. What is remarkable is that in the crystal the neighbor- ization factors; the correction for the anisotropy of absorp- ing molecules together with water molecules form extended tion (,u = 8.99 cm-' for GAV and 8.66 cm-' for GAL) was helices similar to a right-handed a-helix (Figs. 1-3, Table 1). carried out using three reflections at X 900 (the minimum, The helices are all amphipathic. One side of the helix is maximum, and average transmission factors are, respec- hydrophobic and involves the side chains; the other side tively, 41%, 93%, and 68% for GAV and 66%, 99%, and 84% for GAL). The structures were solved by direct methods tAtomic coordinates have been deposited with the Cambridge Struc- using SHELXS-86 (25) and refined by full-matrix least-squares tural Data Base, Cambridge Crystallographic Center, Cambridge, methods using the ENRAF-NONIUS structure determination CB2 lEW, U.K. Downloaded by guest on September 28, 2021 Biophysics: Parthasarathy et al. Proc. Natl. Acad. Sci. USA 87 (1990) 873 b

a

FIG. 2. GAL. (a) Stereoview of the helical turn stabilized by OWL. Note that OW1 is in the plane of the C00- group. (b) Helix formed by GAL in the crystal. This helix is nearly identical to that of GAV in Fig. 1. The helices formed by GAV and GAL differ from those of GAF and GGV; those formed by GAV and GAL have three water molecules stabilizing the helix but the helices formed by GAF (Fig. 3) and GGV have only two water molecules playing a similar role. involves the bound water molecules and hydrogen bonding (i) the increasing hydropathy of the trimer sequence; (ii) the and is hydrophilic. role of OW1 in nucleating/stabilizing the helical conforma- Three factors seem to be important for the formation ofthe tion; and (iii) the role of OWl, OW2 (and OW3) in extending crystalline helix and are discussed in the following sections: the helix. Since there are only three residues in the oligomer, it cannot form a complete turn of the a-helix. One of the characteristics of an a-helix is that the rise per turn is 5.4 A and it needs 3.6 residues to complete a turn. These tripeptides have 1 less residue and lack one CH2-CO fragment to

Table 1. Torsional angles in other hydrated tripeptides in the crystalline state Tripeptide 41 42 4)2 43 4)31 4132 L-Ala-Gly-Gly 159.9 -83.1 169.4 170.9 -6.4 175.2 D-Val-L-Tyr-L-Val -166.5 -83.3 145.6 -77.5 52.2 129.5 DL-Gly-Phe-Gly 127.6 -126.2 131.9 84.3 -4.5 179.2 L-LeU-L-Pro-Gly 152.2 -66.7 160.6 -175.7 -2.6 179.6 L-Tyr-Gly-Gly 163.8 80.7 11.7 -103.8 31.4-152.5 L-Ala-L-Ala-L-Ala* 152.7 -145.7 145.4 -146.9 -9.6 172.2 L-Ala-L-Ala-L-Ala* 162.2 -156.2 149.9 -159.8 -40.0 143.9 Gly-L-Leu-L-Tyr* 170 -131 148 -146 -11 169 Gly-L-Leu-L-Tyr* 154 -146 134 -140 -9 171 Gly-L-Phe-L-Phe -161.9 -126.1 -55.6 -77.7 -20.1 162.6 a-Helical Gly-L-Ala-L-Phe -147.8 -71.2 -33.4 -78.3 -43.3 138.1 Gly-Gly-L-Val -155.8 -77.0 -22.2 -81.1 -50.7 129.0 Gly-L-Ala-L-Val -150.9 -68.2 -38.3 -74.8 -45.6 135.6 Gly-L-Ala-L-Leu -150.4 -67.6 -39.3 -71.3 -45.2 136.9 The 4)31 and 4)32 values have been rearranged to correspond to values observed in the present study. Except for the last two compounds that correspond to the present study and for L- Gly-L-Leu-L-Tyr from ref. 27, the torsion angle values are from table FIG. 3. Stereoview of the helix formed by GAF in the crystal for IV ofref. 22. The asterisk indicates two molecules in the asymmetric comparison with Figs. 1 and 2. unit of the crystal. Downloaded by guest on September 28, 2021 874 Biophysics: Parthasarathy et al. Proc. Natl. Acad. Sci. USA 87 (1990) complete one full a-helical turn. If this peptide were longer, not only assume a helical conformation in the solid state but the location of 03'1 would be taken up by an amino group of also are able to extend the "helix" with just two or three the next residue in the chain. Since these are only tripeptides, water molecules. This fact suggests that such sequences the amino group of the ith residue is linked to 03'1 ofthe last might be capable of forming a part of nucleation in the initial residue by a water (OW1) bridge. The molecule is held in this formation of an a-helix. The importance of sequence is near a-helical conformation by this crucial water bridge obvious from a study of Table 1. Though there are many connecting the NH' and COO- groups (Figs. 1 and 2) tripeptides characterized by crystal structure analysis, they through OWL. OW1 is in the plane of the terminal carboxyl do not form any particular pattern except for the last four group and acts as the fourth residue needed to complete the peptides in Table 1. All these peptides have three common turn by forming two hydrogen bonds, one to N1 ofthe residue factors: (i) the hydrophobicity [as judged from the hydro- i and to the COO- group ofthe residue i + 3, thereby holding pathic indices of Kyte and Doolittle (29)] increases as one the oligomer in the near a-helix conformation. moves from the N terminus to the C terminus, (ii) the The extension of the helix throughout the crystal takes N-terminal residue is glycine, and (iii) the peptides are place by connecting translationally related molecules along a zwitterions. The peptide Gly-L-Phe-L-Phe has this character- cell edge that has a spacing near to 6.0 A by using one more istic pattern of hydrophobicity, but only one of its residues (in GAF and GGV) or two more (in GAV and GAL) water assumes the near a-helical conformation. Other related and molecules (Table 2). The water molecule OW2 in GAF (Fig. longer sequences must be synthesized to find out what 3) and GGV, and OW3 along with OW2 in GAV and GAL, as additional factors are needed to generate longer a-helices in well as OW1, provide the i <- i + 4 type of hydrogen bond solution and in the crystal. typical of the a-helix; in addition, the CO(i + 1) forms a It has been suggested (19) that the removal of the charges C=O...H-N hydrogen bond characteristic of a-helix with on the a-NH' and a-COO- groups by pH titration stabilizes the N-H of a translated i + 2 residue. the a-helix; in spite of the presence of these charges, the Comparison of the Tripeptide Crystal Structures. GAF and trimer sequence forms a helical fold. When any of the above GGV form closely similar crystal structures. GAV has the five sequences forms a part of a polypeptide, there are no same space group as GAF and GGV (Table 2), but the charges, and what effect it has on folding remains to be dimensions of the a and b axes have interchanged. Conse- evaluated. It is also known that glycine occurs preferentially quently, the orientation ofthe helix with respect to the 2-fold at the carboxyl ends (C cap), terminating helices (17). The screw axis is quite different. GAL has a totally different above arguments, which are statistical, in nature suggest that crystal symmetry. In all ofthese structures, the cell edge that a glycine at the amino end of a helix is not preferable; is close to 6 A is parallel to the helix axis. The cell edge that however, they do not rule out the occurrence ofglycine at the is close to 8 A is nearly perpendicular to the helix axis and amino end of a helix in any particular situation. Further, determines the contact regions of the hydrophilic part of glycine as N cap (17) is rather frequent. To give a specific adjacent helices in the crystal. The third cell edge varies example, consider melittin (30), where residues 1-10 form an considerably, depending on the side chains and symmetry. a-helix; the residue 1 starting the helix is actually glycine. Thus, it is seen (Table 2) that these tripeptides are not Two other situations where water molecules interact with isomorphous in their crystal structure but take up different the backbones of helices were discovered independently by space groups and symmetry and yet form the same typical Karle and coworkers (31, 32) and Sundaralingam and co- helix stabilized by water molecules; thus, the folded confor- workers (16, 33). It has been found in solvated a-amino- mation similar to an a-helix is a characteristic property of isobutyric acid-containing hydrophobic peptides that a water these sequence-related tripeptides. Whether this property is molecule is inserted into the helical backbone (31, 32), reflected when they are a part ofa longer polypeptide remains resulting in a distortion of the helical backbone and gener- to be tested by synthesizing longer oligomers. ating an amphiphilic character to the helix containing only hydrophobic sequences, quite similar to the helices discussed DISCUSSION in this manuscript. A similar solvation of a helix, but of hydrophilic residues, occurs in troponin C and has led The formation of a-helices in disordered polypeptides is a recently to an analysis of the role of water molecules in the classical nucleation event (28). The rate-limiting step is the folding/unfolding ofa-helices in protein structures using data formation of the first segment with an equilibrium constant from the Protein Data Bank (33); the hydrated segments typically in the order of io-4. At least four residues in helical display a variety of reverse turn conformations that may be conformation are necessary to form one turn at first; addition considered as folding intermediates involved in the folding/ of amino acids to either end of the helix is rapid with the unfolding of a-helices. The occurrence of such hydrated equilibrium constant close to 1. The five tripeptide molecules helices or turns in very different situations indicates their relative stability. One major difference exists between the Table 2. Unit cell, space group, and cell contents solvated helices discussed above and the crystalline helices Peptide GAF GGV GAV GAL of the tripeptides; in the former, a helix seems to be inter- a, A 5.879 (1) 5.786 (1) 8.056 (1) 6.023 (1) rupted, whereas in the latter, a continuous helix in the crystal b, A 7.966 (1) 7.954 (2) 6.032 (1) 8.171 (1) is generated because of the interaction with water. c, A 17.754 (2) 14.420 (3) 15.779 (1) 32.791 (2) In the process of refolding of reduced polypeptides, inter- (3 95.14 (2) 93.85 (2) 98.52 (1) actions among nearest-neighbor side chains are thought to S.G. P21 P21 P21 P212121 trigger the formation of nucleation sites. The knowledge of Z 2 2 2 4 intermediate conformations adopted by these nucleation sites Water Mol. 2 2 3 3 is important in further understanding such nearest-neighbor R factor 0.031 0.040 0.067 0.051 interactions. Since protein folding is hydrophobically driven, of and is an essential con- a, b, c, and ,B are the unit cell parameters. S.G. refers to space hydration polar nonpolar groups group. The structure of GAF is reported in ref. 22; that of GGV is formational determinant for oligo- as well as polypeptides. reported in ref. 23 and reinterpreted by us in ref. 22. The results of Our studies here provide a graphic method of studying the our x-ray analyses of GAV and GAL are reported here. Water Mol. relationship of sequences to folding preferences in tripep- refers to the number of water molecules per tripeptide oligomer. The tides; whether these sequence preferences for a-helical con- values in parentheses denote estimated standard deviations. formation seen in tripeptides are maintained in longer pep- Downloaded by guest on September 28, 2021 Biophysics: Parthasarathy et al. Proc. Natl. Acad. Sci. USA 87 (1990) 875 tides (containing these tripeptide consequences) depends on 15. Marqusee, S. & Baldwin, R. L. (1987) Proc. Natl. Acad. Sci. the guest-host interaction and remains to be explored. USA 84, 8898-8902. 16. Sundaralingam, M., Drendel, W. & Greaser, M. (1985) Proc. Note Added in Proof. X-ray crystallographic analysis ofGAI has been Natl. Acad. Sci. USA 82, 7944-7947. completed and it also shows that GAI forms a helix very similar to 17. Richardson, J. S. & Richardson, D. C. (1988) Science 240, GAV. 1648-1652. 18. Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641. We are grateful to Drs. G. Markus, G. Tritsch, G. B. Chheda, N. 19. Fairman, R., Shoemaker, K. R., York, E. J., Stewart, M. & Ramasubbu, and K. Bhandary for reviewing earlier drafts and Baldwin, R. L. (1989) Proteins Struct. Funct. Genet. 5, 1-7. making valuable suggestions. This work was supported in part by 20. DeGrado, W. F., Wasserman, Z. R. & Lear, J. D. (1989) National Institutes of Health Grants GM 24864 and CA 23704. Science 243, 622-628. 21. Regan, L. & DeGrado, W. F. 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International Tablesfor Crystallography (1974) (Kynoch, Bir- 8. Epand, N. H. & Scheraga, H. A. (1968) Biochemistry 8, 2864- mingham; distributed by Kluwer, Dordrecht, The Nether- 2872. lands), Vol. 4. 9. Taniuchi, H. & Anfinsen, C. B. (1969) J. Biol. Chem. 244, 27. Subramanian, E. & Parthasarathy, R. (1987) Curr. Sci. (India) 3864-3875. 56, 1210-1213. 10. Kabsch, W. & Sander, C. (1984) Proc. Nati. Acad. Sci. USA 28. Creighton, T. E. (1987) Nature (London) 326, 547-548. 81, 1075-1084. 29. Kyte, J. & Doolittle, R. F. (1982) J. Biol. Chem. 157, 105-132. 11. Zimm, B. H. & Bragg, J. K. (1959) J. Chem. Phys. 31, 526-535. 30. Terwilliger, T. C. & Eisenberg, D. (1982) J. Biol. Chem. 257, 12. Bierzynski, A., Kim, P. S. & Baldwin, R. L. (1982) Proc. Nati. 6016-6022. Acad. Sci. USA 79, 2470-2474. 31. Karle, I. L., Flippen-Anderson, J. L., Uma, K. & Balaram, P. 13. Dyson, J., Cross, K. J., Houghton, R. A., Wilson, I. A., (1989) Biopolymers 28, 773-781. Wright, P. E. & Lerner, R. A. (1985) Nature (London) 318, 32. Karle, I. L., Flippen-Anderson, J. L., Uma, K. & Balaram, P. 480-483. (1988) Proc. Natl. Acad. Sci. USA 85, 299-303. 14. Shoemaker, K. R., Kim, P. S., York, E. J., Stewart, J. M. & 33. Sundaralingam, M. & Sekharudu, Y. C. (1989) Science 244, Baldwin, R. L. (1987) Nature (London) 326, 563-567. 1333-1337. Downloaded by guest on September 28, 2021