Peptide Models 6. New @-Turn Conformations from ab Initio Calculations Confirmed by X-ray Data s f ProteinsL Andrhs Perczel,t** Michael A. McAllister,t Phl Cshszhr,t*g and Imre G. Csizmadia'*+ Contribution from the Department of Chemistry, University of Toronto, Ontario, Canada M5S 1 AI , Department of Organic Chemistry, Eat& University, Budapest 11 2 POB 32 H-1518. Hungary, and Apotex Inc., 150 Signet Drive, Weston, Ontario, Canada M9L 1 T9 Received May 20, 1992 Abstract: In an attempt to determine intrinsically stable hairpin geometries, a number of triamide conformations of For-Ala-Ala-NH2 were investigated using ab initio calculations (HF/3-21 G). Previous ab initio calculations of selected diamides of single amino acid residues (e.g., For-Ala-NH2) suggested that the a,-type backbone conformation (6 = -54', +=-45') is not a minimal energy structure, although in globular proteins the (aL),, units (referred to as a-helices) are the most frequently found conformations. The lack of the aL conformation made the application of ab initio calculations in peptide geometry analyses questionable. In contrast, for triamides (e.g., For-Ala-Ala-NH2) the appearance of the aL backbone subconformation is confirmed in the a,6, conformation (usually referred to as type I &turn). This intrinsically stable conformation is the most frequently found hairpin structure in proteins. The existence of the eL conformation (4 =d o o , +=120') in chiral diamides (such as For-Ala-NH2, For-Ser-NH2, or For-Val-NH2) has never been confirmed by ab initio studies, although X-ray analyses of proteins revealed the existence of the polyproline I1 conformation [(eL),,] a long time ago. The herein presented stable Y ~ E ~ and bDeL hairpin conformations, calculated by abinitiomethods, legitimize the"missing" e, backbonegeometry. The fact that somelegitimate backboneconformations (a, and e,) appear only in triamides and not in diamide systems assigns a specific role to triamide models in understanding protein conformations. The importance of some triamide conformations, especially type I and type I1 &turns, is emphasized. This study summarizes all the possible [18 (30) conformations depending on the d or T "selection rule"] hairpin geometries determined for For-Ala-Ala-NH2 using ab initio computations. We were able to identify all 30 ab initio yielded conformations as backbone substructures of globular proteins, determined by X-ray crystallography. The 30 optimized triamide structures present a unique opportunity to understand the conformational behavior of @-turns @bends or hairpins). This may have far-reaching consequences in understanding the j3-turn-mediated protein folding. Introduction Besides the two major secondary structural elements'-) [the a-helix (& =-60 f 30, qi =-60 f 30),, and the 8-pleated sheet (& =-150' f 30, +i =150' f 30),], the third most frequently found4J structural unit in globular proteins is the @-turn conformation.G8 Smith and Pease* reviewed in detail the reverse turns in peptides and proteins. Reverse turns, hairpins, 8-bends, or &turns are structural elements consisting of four successive amino acid residues (labeled 1,2,3, and 4) at positions i, i +1, i +2, and i +3 in proteins. The variety of definitions suggested in the past quarter of a century clearly illustrates the evolution of the &turn concept. Adhering to the original definitions of Venkatachalam: &turns are classified into conformational types by their values of cj i +, , +i +l , &+I, and +i+2 torsional angles. On the basis of the four backbone torsional angle values of the second * Address correspondence to this author. Current address: Laboratoire de Chime Thorique, UniversitC Nancy 1 C.N.R.S., 54506 Vandoeuore-les- Nancy, France. +University of Toronto. 5 Apotex Inc. 1 Dedicated to the memory of Professor M. KajtBr, the late virtuoso of stereochemistry. (1) Pauling, L.; Corey, R. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 729- 740. (2) Pauling, L.; Corey, R.; Branson, H. Proc. Natl. Acad. Sci. U.S.A. (3) Levitt, M.; Chothia, C. Nature (London) 1976, 261, 552-558. (4) Crawford, J . L.; Lipscomb, W. N.;Schellman,C. G. Proc. Natl. Acud. (5) Zimmerman, S . S.; Scheraga, H. A. Proc. Narl. Acad. Sci. U.S.A. (6) Venkatachalam, C. Biopolymers 1968, 6, 1425-1436. (7) Sibanda, B. L.; Thorton, J . M. Nurure. (London) 1985,316,170-174. (8) Smith, J .; Pease, L. Crit. Reu. Biochem. 1980, 8, 315-399. Edtvijs University. 1951, 37, 205-21 1. Sci. U.S.A. 1973, 70, 538-542. 1977, 74, 41264129. and third residues, there are three major types of folded conformations: I, 11, and I11 &turns (Chart I). About a decade later, in the analysis of the @turn content of globular proteins, a distance criteria was also i ntrod~ced.~~.~ Accordingly, the Cy - Ci distance must be shorter than 7 A. Often an intramolecular H-bond can befound in @-turns, where the NH of the i +3 residue points toward the carbonyl oxygen of the ith residue (1 - 4-type H-bond) as shown in Figure 1. Although this 1 - 4 hydrogen bond has never been proved to be a necessary condition for @-turns, it is frequently found in peptides and proteins on the basis of X-ray9aqc and NMR structure determinations,"J and therefore a misconception has developed over the years that such a hydrogen bond is an essential structural featureof 0-turns. This H-bond pattern was also used as a criterion for @-turn assignment in proteins.9a~c The Vankatachalam6-predicted +i +2 =0' value for several types of &turns was not satisfied in several structure assignments in globular proteins. Consequently, Chou and Fa~man~bsuggested a larger tolerance for the +i+2 torsional angle (-50' I +1+2 I 50'). More recently, Wilmot and Thorton" demonstrated that the 1c;+z =0' criterion is one of the reasons that numberous turns are identified as "distorted." The experimental value of $i +2 is often around 45' or -45'. This finding is in perfect agreement with our previous analysisl2J3 of ab initio Ramachandran maps, (9) (a) Levitt, M.J . Mol. Biol. 1976,104,59-107. (b) Chou, P. Y.; Fasman, G. D. J. Mol. Biol. 1977, 115, 135-175. (c) Kabsch, W.; Sander, C. Biopolymers 1983, 22, 2577-2637. (IO) Dyson, H. J .; Rance, M.; Houghten, R. A.; Lerner, R. A.; Wright, P. A. J. Mol. Biol. 1988, 201, 161-200. (11) Wilmot, C. M.; Thorton, J . M. Protein Eng. 1990, 3, 479493. (12) Perczel, A,; Angyan, J . G.; Kajtar, M.; Viviani, W.; Rivail, J .-L.; Marcoccia, J .-F.; Csizmadia, I. G. J. Am. Chem. SOC. 1991,113,62564265. OOO2-7863/93/1515-4849$04.00/0 0 1993 American Chemical Society 4850 J. Am. Chem. Soc., Vol. 115, No. 11, 1993 Perczel et al. 0 dMODEL g dCRl l l CAL < A Figure 1. (a) Sequenceof four successiveamino acid residues forming a &turn backboneconformation. (b) A schematic representation of the 1 - 4 hydrogen bond in a &turn structure and the 7-A upper limit of the critical distance (&, =d) whichassigns the secondary structural elements according to the "classical" definition. Inmodel compounds (e.g., For-Ala-Ala-NH2) the model distance (&del) isalways shorter than the critical distance for a peptide with real Ci' and Cy atoms (c). chart I +i,l *i+2 topological code TYPE I -60 -30 -90 0 a a 0 c a L L aL'L aL6L L D ' L I D ' LdD X I -60 120 80 1 x 1 -60 -30 -60 -30 aLaL chart n H2 02 H CH3 H4 I II \ / I N2 N4 / \ I II H3 03 H CH3 ll 01 where none of the nine conformational minima had +values around 0" while values around - 4 5 O (aL, tSD, or r,,) and around 45" (ao, tSL, or T~ conformations) were found. Therefore, we propose to distinguishI3 the three different forms of type I and type I1 &turns on the basis of +i +z. These conformations are identified by the topological codes given in Chart I. The type I11 &turn is determined as a single turn of a 3 helix and has therefore a single topological code only. The smallest N- and C-terminal protected @-turn model, incorporating two chiral amino acids, has the structure shown in Chart 11. In an N-formyl dipeptidamide model, the two amino acids represent the second and the third residues of a &turn (cf. Figure 1). In this model the Hl-C'l bond of the N-terminal formyl group is shorter by -0.5 A and the N4-H4* bond length of the C-terminal amide group is shorter by 0.6 A, as compared to the corresponding Cp- C'1 and the N4 - CP,4 bonds. Due to this shortening, the distances dmdcl and may differ from each other by no more than 0.6 A +0.5 A =1.1 A, depending on the orientation of the two bonds (Figure 1). Although the classification of 8-turns is traditionally made on the basis of the backbone torsional anglevalues (4i , +i ), the degree (13) Perczel, A.; Kajtar, M.; Marcoccia, J.-F.; Csizmadia, I. G. J. Mol. Srrucr. 1991, 232, 291-319. Figure 2. (Left) Hairpin conformation of a polypeptidechain. A schematic illustration of anuntwisted &turn and a backbonetwisted by T degrees is shownonthe right. b"L b"L Figure 3. yLyL conformation of For-L-Ala-L-Ala-NHz corresponding to a rather twisted backbonegeometry ( T ~ ~ + : - ~ ~ + =167.8'). Table I. Different Types of &Turnsa in Peptides Identified on the Basis of Experimental Studies6,8J I type 41 $1 $2 $2 new code(s)b I -60 -30 -90 0 a L a L , ~ L Y L ~ I' 60 30 90 0 ~ D ~ D , ~ D Y D , 4, I1 -60 120 80 0 c L ~ D , ~ L Y D . 11' 60 -120 -80 0 W Y L ~ cDYL, I11 -60 -30 -60 -30 QLCYL 111' 60 30 60 30 aDaD IV ambiguously defined V ambiguously defined VIa -60 120 -90 0 ( L aL , ~ L Y L , VIb -120 120 -60 0 B L ~ L ~ B ~ Y L , ~ L ~ ~ VI11 -60 -30 -120 120 C& VI1 ambiguously defined "Only types I, 11, and I11havemore than sporadic Occurrencein globular proteins according to X-ray studies. Indegrees. of folding or unfolding of a @-turn can be defined in a simpler way, on the basis of the twisting of the hairpin conformation. In agreement with the pioneering work of Levitt? wehave recently introducedI4 the CY - CP,, - C: , - CK3 torsional angle labeled as T (see Figure 2) that describes the overall angularity of the backbone conformation with values -180" I T I 180". The global minimum (the yLr L conformation), for example, has T?L?L = 168.4', which can quantitatively describe the degree of unfolding (Figure 3). Considering the criterion that the Cy - C: distance must be shorter than 7 A, only a fraction of the @-turn conformations (-90" I T I 90") can be assigned as such. For the first threeoftheeight different types (I-VIII) ofb-turns (Table I), their mirror image conformations (types 1', 11', and 111' &turns) have also been suggested previously.6 Although both the type I @-turn and the type I' @-turn conformations incorporate only L-amino acids, they have conformationally (14) McAllister, M. A.; Perczel, A.; Csaszar, P.; Csizmadia, I. G. J. Mol. Szrucr., in press. 8- Turn Conformations Confirmed by Protein X-ray Data chart m 01 H R H3 -300 40 8~ TL J. Am. Chem. SOC., Vol. 11.5, No. 11, 1993 4851 do 8L YL I I I I I enantiomeric peptide backbones.i2 Therefore, the conformational enantiomer of the aLuL conformation (type I [or 1111&turn) is the a,u, conformation labeled traditionally as the type I [or 1111&turn. (Duplications reported in Table I originate from the historic evolution of the concept; both the type I and the type I11 &turns have an aLaL-like backbone geometry, but these were derived from different sources and therefore are labeled differ- ently.) The extraction of the remaining &turn conformations, fulfilling the angularity (-90 5 7 I 90) or distance (d I 7 A) criteria, is hardly possible on the basis of earlier approaches. Even if more &turn conformations exist than can be detected by analyzing a 4D-RamachandranI3-type potential energy hyper- surface (PEHS), these structures, due to the rate Occurrence of these conformations in globular proteins, cannot be extracted from the X-ray data analyses of these proteins. Multidimensional conformational analysis (MDCA)I5 pre- dicted2 nine minima on the full Ramachandran map,16 E = E(&#), where the torsional angles are defined according to IUPAC-IUB17 convention as shown in Chart 111. (Let us assume thatw, andw2areconstants;usuallywl andw2=180 or sometimes O O ) . Each of these nine minima represents the only energy minimum in a given catchment region.I8 Figure 4 shows the Occurrence of the nine minima (aL, aD, BL, yu yo, 6L, 6,, eL, e,) in an idealized fashion. It should benoted that according to the IUPAC-IUB conventioni7 the 4 and the # values vary between -180 and 180O. This domain is indicated by the dashed square in the center of Figure 4. For various reas0ns,12.~3 during conformational analysis it is convenient to use a different cut of the same potential energy surface (PES), namely any of the four identical quadrants encircled by the solid lines in Figure 4. With this choice of representation, the spatial arrangement of the nine minima may be specified as shown in Chart IV. Torsional angles and #TOP span the range from Oo to 360 in accordance with the earlier suggestion,I2 while in the IUPAC-IUB ~nventi on ~ these 4 and $ variables vary from During the conformational analysis of PCONH-CHK-CON- HQ using ab initio cal c~l ati ons,~~-~~ the absence of the aL and (15) (a) Csizmadia, 1. G. Generaland theoreticalaspectsof the thiolgroup. In The Chemistry of functional groups. The chemistry of the thiol group; Patai. S., Ed.; Wiley and Sons: New York, 1974; pp 1-109 (particularly pages 36-41 including Figures 23 and 24 as well as Table 20). (b) Bertran, J. Multidimensional Theoretical Stereochemistry and Conformational Potential Energy Surface Topology. In New Theoretical Concept for Understanding Organic Reactiorw;Csizmadia, I. G. Ed.; D. Reidel Publishing Co.: Dordrecht, 1989: BD 1-31. -180 to 180. r r - .. _ _ , (16) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. J. Mol. (17) IUPAC-IUB Commission on Biochemical Nomenclature. Biochem- Bi d. 1963, 7, 95-98. istry 1970, 9, 3471-3479. (18) Potential Energy Hypersurfaces; Mezey, P. G. Ed.; Elsevier Science .- - Publishers: Essex, 198-j. (19) (a) Sellers, H. L.; Schafer, L. J . Am. Chem. SOC. 1978,100, 7728- 7729. (b) Schafer, L.; Sellers, H. L.; Lovas, F. J.; Suenram, R. D. J. Am. Chem. Soc. 1980,102,6566-6568. (c) Schafer, L.; Van Alsenoy, C.; Scarsdale, J. N. J . Chem. Phys. 1982,76,1439-1444. (d) Klimkowski, V. J.; Schafer, L.; Momanay, F. A.; Van Alsenoy, C. J. Mol. Srruct. 1985, 124, 143-165. (e)Scarsdale, J. N.; Van Alsenoy,C.; Klimkowski, V. J .; Schafer.L.; Momany, F. A. J. Am. Chem. SOC. 1983,105,3438-3445. (f) Schafer, L.; Klimkowski, V. J.; Momany, F. A.; Chuman. H.; Van Alsenoy. C. Biopolymers 1984,23, (20) Viviani, W.; Rivail, J.-L.; Perczel, A.; Csizmadia, I. G. J. Am. Chem. SOC., submited for publication. 2335-2347. - 240t eL peptide conformations was noted. By the calculation of several dozens of relaxed grid points, the shape of the PES for For- Ala-NH2 was recently analyzed in the vicinity of the uL conformation, but no minimum was f0und.2~Nevertheless, the shapeof thePES, in thearea where thea,conformationisexpected to be, suggests that even a minor stabilizing force (such as a hydrogen bond) could result in a local minimum, giving legitimacy to the aL backbone structure. To the best of our knowledge, various experimental methods, including X-ray crystallography, have never identified the uL backbone conformation for any single amino acid diamides, which agrees perfectly with the ab initio results. Nevertheless, each of the nine minima specified in Chart IV are legitimate both in terms of multidimensional conforma- tional analysis and on the basis of X-ray-analyzed structures in larger peptides and pr0teins.2~9~~ (The conformational oligomers (u& and (e,),,, the so-called a-helix and poly-L-proline I1 secondary structural units, Occur frequently in globular proteins.) However, the absence of the mentioned minima (aL and e,) in the (21) (a) Head-Gordon, T.; Head-Gordon, M.; Frish, M. J.; Brooks, C., 11; Pople, J. A.; In?. J. Quantum Chem. Quantum Biol. Symp. 1989, 16, 311- 319. (b) Head-Gordon, T.; Head-Gordon, M.; Frish, M. J.; Brooks, C., 11; Pople, J. A.; J. Am. Chem. Soc. 1991,113, 5989-5997. (22) Bohm, H.-J.; Brode, S. J. Am. Chem. SOC. 1991, 113, 7129-7135. (23) Bertran, J. Peptide conformational Potential Energy Surfaces and their relevance to protein folding. In Molecular aspect of biotechnology: computational models and theories; Perczel, A., Viviani, W.. Csizmadia, I. G., Eds.; Kluwer Academic Publishers Co.: Dordrecht, 1992; pp 39-82. (24) McAllister, M. A,; Perczel, A.; Csaszar, P.; Vladia, W.; Rivail, J.-L.; Csizmadia, I. G. J. Mol. Srruct., in press. (25) (a) Aubry, A.; Marraud, M.; Protas, J .; Nccl, J. C. R. Acad. Sci. Paris 1974, 287c, 163-166. (b) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad. Sci. Paris 1973,276~, 1089-1092. (c) Aubry, A. These pour Docteur de Sciences Physiques, Universite de Nancy, France, 1976,102-107. (d) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad. Sci. Paris 1971, 273c, 959-961. (e) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad. Sci. Paris l974,287c, 163-166. (f) Aubry, A.; Cung, M. T.; Marraud, M. Cryst. Srruct. Commun. 1982,II, 129-133. (g) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad. Sci. Paris 1974,287~ 697-700. (b) Aubry, A.; Marraud, M.; Cung, M. T.; Protas, J. C. R. Acad. Sci. Paris 1985,280~. 861-863. (i) Aubry, A,; Protas, J.; Marraud, M. Acra Crysfallogr. 1977, 833, 2534-2539. (j) Aubry, A.; Protas, J .; Marraud, M.; Neel, J. Acta Crystallogr. 1976, 832, 2749-2759. 4852 J. Am. Chem. SOC., Vol. 115, No. 11, 1993 Perczel et a/. Table 11. Experimental Conformation of Selected Single Amino Acid Diamides Obtained from X-ray Crystalloaraphy amino confor- acid N-terminal C-terminal QP + mation ref MeCO- PrCO- MeCO- MeCO- MeCO- MeCO- MeCO- BuCO- BuCO- BuCO- -NHiPr -NHiPr -NHiPr -NMe2 -NMe2 -NMe2 -NHEt -NHEt -NHMe -NHMe -78 160 CL -1 12 142 tL -169 175 B L -1 26 162 B L -92(-90) 123(122) CL -132 77 6, -9 1 144 tL -9 1 144 CL -72 165 CL -92 151 EL 25a 25b 25c 25d 25e 25f 2% 25h 25i 25j In degrees. There were two molecules of different geometries in the unit cell. Side chain torsional angles are xI =70, x2 =88. I + Figure 5. Illustration of the Grand Canyon region of a 2D- Ramachandran map that includes the BL, yL, et, and 6, conformations. The idealized conformations are denoted by open stars, while the arrows indicate the approximate shifts of the ideal conformation to the actual ones. simplest peptide model (PCONH-CHR-CONHQ) could have far-reaching consequences, resulting in the conclusion that polypeptide backbone conformations cannot be modeled using PCONH-CHR-CONHQ-type models. This would mean that, from a conformational point of view, proteins cannot besimply regarded as the polymers of -CONH-CHR-CONH-systems but must beconsidered as built from larger substructures. According to selected crystallographic data, amino acid diamides adopt minimal energy conformations (Table 11) close to cL, Y ~ , and 6, on the E =E(4, $) PES. These three minima are also close to each other geometrically. These crystallographically determined minima are located in a common region of a Grand Canyon (as shown in Figure 5) that has been constructedz3 using Poples ab initio energy contour diagram.2 Type I and I1 @-turns incorporate the aL and tL conformations at the second [or ( i + l)th] position of the hairpin conformation (see Table I). These (26) (a) Aubry, A.; Marraud. M.; Protas, J . C. R. Acad. Sci. Paris 1975, ZSOC, 509-512. (b) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1977,833,2399-2406. (c) McLarfi, M.; Aubry, A.; Marraud, M. Eur. Biophys. J. 1986,14,43-51. (d) Boussard, G.; Marraud, M.; Aubry, A. Int. J. Pept. Protein Res. 1986, 28, 508-517. (e) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1979,835,694-699. ( f ) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1980,836, 321- 326. (g) Aubry, A.; Boussard, G.; Marraud, M. Acta Crystallogr. 1981,837, 1474-1477. (h) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1980,836,2822-2824. (i) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1980,836,2825-2827. (j) Aubry, A,; Lecomte, C.; Boussard, G.; Marraud, M. J. Chim. Phys. Phys. Chim. Biol. 1983.80, 609-614. (k) Aubry, A.; Marraud, M. Acta Crystallogr. 1985, C41,65-67. (I ) Aubry, A.; Vitoux, B.; Marraud, M. Biopolymers 1985, 24, 1089-1 100. (m) Aubry, A.; Cungt, M. T.; Marraud, M. J. Am. Chem. SOC. 1985, 107, 7640-7647. (n) Aubry, A.; Ghermani, N.; Marraud, M. Int. J. Pept. Protein Rb. 1984, 23, 113-122. ( 0) Milner-White, E. J .; Ross, B. M.; Ismail, R.; Belhadj-Mostefa, K.; Poet, R. J. Mol. Biol. 1988,204,777-782. (p) Boussard, G. Marraud, M.; Aubry, A. Biopolymers 1979,18,1297-1331. (9) Boussard, G.;Marraud,M. J. Am. Chem.Soc. 1985,107,1825-1828. (r)Liang,G.-B.; Rito, C. J .; Gellman, S. H. J. Am. Chem. SOC. 1992, 114, 44404442. backbone geometries were not obtained from ab initio studies of chiral diamide model ~I ~- ~~ (e.g., For-Ala-NHz or For-Val-NH2). The conformational enantiomers of these @-turns, the type I and the type 11 structures, incorporate not the unstable CY, and t L subconformations but the stable aD and tD backbone orientations. Several papers have been published9- I detailing results obtained by using theoretical predictions and statistical analyses of @-turn distribution frequencies in globular proteins. However, the question of whether &turns may be considered not only as a recognizable secondary structural element in proteins9 but also as intrinsically stable conformational elements of simple triamides has still not been answered. Although experimental evidence accumulated from studies of model compounds (e.g., -Pro-Xxx-) in apolar such as CC4, suggests that @-turn conformations are the most enthalpically favored patterns for several small peptides, the lack of gas-phase evidence may suggest the following alternatives. It is quite possible that @-turn conformations exist only in an environment created by solvation, long-range interactions, intermolecular H-bonds, etc. On the other hand, 8-turns may be intrinsically stable structures and they may exist even in a vacuumwithout the stabilizing interaction of any side chain or environmental effect. TheX-ray crystallography of dipeptidederivatives (e.g., PCO- Xxx-Yyy-NHQ) having a hairpingeometry (seeTable 111) shows that the folded conformation is always stabilized by intermolecular and/or environmental In solution the stabilizing effect of solvents can never be excluded. Since gas-phase data on tripeptide structures are currently not available, there are no experimental data which prove or disprove the intrinsic stability of @-turns. In the 1970s, Scheraga and others investigated @-turn conformations using molecular mechanics cal cul ati ~ns.~~ The structural analysis of Ac-Xxx-Yyy-NHMe-type peptides resulted in useful geometrical data, although these force-field calculations cannot answer the question of existence or nonexistence due to the experimental origin of the parameters used. For example, force-field calculations (e.g., ECEPP/2) for Ac-L-Ala-NHMe resulted in a stable aL structure that must be regarded as an artifact in view of recent ab initio cal c~l ati ons.~~J ~-Z~ Reliable answers can only beexpected from high-level ab initio calculations. Although some attempts have been made28 to include correlation energy in the case of diamides of single amino aeids, the triamide systems presently remain much too large for such sophisticated calculations. Even at a lower level of theory, only a limited number of computations have been published13,i423.29,30 on selected triamide conformations. The more than two dozen optimized dipeptide diamide geometries reported here should provide a unique opportunity for analysis of the conformational behavior of @-turns, which hopefully will lead to a more accurate understanding of protein 3D-structures. Computational Methods The fully relaxed minimal energy conformations were calculated using full geometry optimizations by gradient methods with the GAUSSIAN 90 program3 using the 3-21G basis seP2 on a Cray X-MP/28 supercomputer. This work is exploratory in its nature, and calculations using a larger basis set at the HF or MP level of theory may beperformed (27) (a) Zimmerman, S. S.; Scheraga, H. A. Biopolymers G77,16,81L 843. (b) Zimmerman, S. S.; Scheraga, H. A. Biopolymers 1978, 17, 1849- 1869. (c) Zimmerman, S. S.;Scheraga, H. A. Biopolymers 1978,17,1871- 1884. (d) Zimmerman, S. S.; Scheraga, H. A. Biopolymers 1978,17,1885- 1890. (28) Frey, R. F.; Coffin, J .; Newton,S. Q.; Ramek, M.; Cheng, V. K. W.; Momany, F. A.; Schafer, J. Am. Chem. Soc. 1992, 114, 5369-5376. (29) Chesnut, D. B.; Phung, C. G. Chem. Phys. Lett. 1991,183,505-509. (30) Sapsa, A.-M.; Daniel, S. B.; Erickson, B. W. Tetrahedron 1988,44, (31) Frisch, M.; Head-Gordon, M.; Trucks, G. W.; Foresman, J . B.; Schelegel, H. B.; Raghavachari, K.; Robb, M. A.; Brinkley, J . S.; Gonzalez, C.; Defrees, D. J .; Foz, D. J .; Stewart, J . J . P.; Topiol, S.; Pople, J . A,; GAUSSIAN 90(Revision F Version); Gaussian Inc.: Pittsburgh, PA, 1990. (32) Binkley, J . S. ; Pople, J . A.; Hehre, W. J . J. Am. Chem. SOC. 1980, 102,939-947. 999-1006. 8- Turn Conformations Confirmed by Protein X-ray Data Table 111. Selected Experimental Conformations of Dipeptide Derivatives Determined by X-ray Crystallography J. Am. Chem. SOC., Vol. 115, No. 11, 1993 4853 amino acids N-terminal C-terminal @ l a *I 42 *2 &turn type conformation ref 26 Pro-Gly BuCO- -NHMe -7 1 157 -76 175 ~ L L a Pro-Ala PrCO- -NHlPr -59 136 66 14 I1 CLQD b Pro+ Ala IPrCO- -NHPr -62 137 96 3 I1 C L ~ D b Pro- Asp BuCO- -NHMe -57 134 59 26 I1 C L ~ D C Pro- Asn BuCO- -NHMe -59 138 66 11 I1 C L ~ D C Pro-Asp(0Me) BuCO- -NHiPr -66 -20 -9 1 6 I C Pro-His BuCO- -NHMe - 63 -22 -7 0 -20 I a L h d D- Ala-Pro BuCO- -NHI Pr 60 -140 -89 9 11 ~ D Y L e D- Ala-D-Pro BuCO- -NHIPr 64 -152 83 -156 WD f Gly-Pro BuCO- -NHMe -79 174 -8 5 -22 VIa C L ~ L g Pro-Gly BuCO- -NHIPr -64 137 84 -3 I1 CLYD h Ala-Pro PrCO- -NHPr -129 76 -67 -22 VIb ha, i Gly-Gly BuCO- -NHPr -69 -25 -8 9 3 I a L a 1 j Ala-Gly BuCO- -NHI Pr -68 132 -8 3 2 VIa C L ~ L j Pro-Thr BuCO- -NHMe -66 -22 -103 7 I ~ L Y L k Pro-Pro BuCO- -NHMe -60 138 -9 5 -7 VIa C L ~ L 1 Pro-D-Pro BuCO- -NHMe -58 134 83 -7 I1 CLYD I Pro-Pro BuCO- -NHMe -60 138 -9 5 -7 VIa C L ~ L m Pro-Cys(Me) BuCO- -NHMe -6 1 132 62 17 I1 C L ~ D m Pro-Phe BuCO- -NHMe -64 139 62 23 I1 f LaD m Pro-Tyr BuCO- -NHMe -59 137 73 9 I1 ~ L Q D m Pro-D-Tyr BuCO- -NHMe -64 137 73 9 I1 C L ~ D m Pro-Ser BuCO- -NHMe -60 -30 -7 5 -1 1 I ( Y L ~L n Pro-D-Ser BuCO- -0Me -64 -29 76 17 a~% n Pro-D-Ser BuCO- -NHMe -59 133 76 8 I1 1 f f D n In degrees. Table IV. Comparison of the Q and $ Torsional Angles Obtained in ab Initio Calculations under Normal and Tight Optimizations on Selected Conformations of For-L-Ala-NHz and For-L-Ala-L-Ala-NH2 torsional anglea compd conformation type normalb tightC For-Ala-NH2 6 Q 63.8 63.8 * 32.7 32.7 + 170.9 170.5 * 67.7 67.3 * 67.0 67.0 * 66.4 66.4 BI 4 -168.4 -168.3 71 Q -84.4 -84.5 For-Ala-Ala-NH2 y, yI Q -84.2 -84.2 Cp -84.9 -85.0 change 0.0 0.0 0.1 0.4 0.1 0.4 0.0 0.0 0.1 0.0 a In degrees. Max force <3 X au. Max force 51 X au. in the future on selected minima. The authors are fully aware of the limitations of the 3-21G basis set, although work by Pople and co-workers21albindicates that in the case of peptides even the 3-21G basis set is sufficient. The initial geometries of the selected triamides were generated on the basis of the previously optimized For-~-Ala-NHz structures.12 The For-~-Al a-~-Al a-NHz geometries were then subse- quently optimized. The final forces along the internal coordinates in the relaxed structures ranged from 2.2 X to 2.1 X 10-4 au, while the value of the root mean square of the forces (rms) was between 5.4 X lo- and 5.7 X au. One may be concerned about the convergence of geometry optimization under these normal conditions. The ultimate test of accuracy, in this particular case, is not so much the magnitude of the residual force but the self-consistency of the 4 and +values as these torsional angles characterize peptide conformations. In order to check this point, normal and tight optimizations have been carried out on selected conformations. As may beseen from Table IV, the absolute value of torsional angle changes, Le., IAq51 and lA+I. are very small. Since the geometry-optimized q5 and * values are needed in the present paper mainly to determine which catchment region a computed conformation belongs to (these characteristic conformations are separated from each other by several tens of degrees), the achieved accuracy is more than adequate (Table IV). The y, yl conformation was found as the global minimum (E[RHF] =-656.963 681 hartrees) and was used as reference point for AE calculations. scope Several different types of criteria (such as distance, torsional angle, or the 1 - 4 hydrogen bonding) have been used to identify Table V. Optimized ab Initio SCF (3-21G) Geometries for Type I &Turn Conformations of For-L-Ala-L-Ala-NH2 initb Wnvc 01 41 *I 42 $2 w3 &del d Tmcdel &rite Tcrit 0 1-HN4 01-*N4 01-NH4-N4 01-HN3 01***N3 01-HN3-N3 02-HN4 02--N4 02-NH4-N4 w2 max force rms force E AE SDV LDBg Q D ~ D ( Y D ~ D 171.2 60.4 28.3 179.0 62.3 24.9 179.1 5.33 -66.9 5.97 -70.8 2.03 3.03 172.4 3.03 3.10 3.06 3.23 91.1 6.5 X 0.956 139 4.73 28 77 -84.5 1.8 x 10-5 ~ D Y D 67, 173.5 62.3 37.0 74.1 174.9 6.06 -79.3 6.64 -80.4 3.76 4.00 97.1 3.20 3.27 1.93 2.83 147.1 1.1 x 10-4 0.950 869 8.04 3 10 -172.9 -58.0 -85.3 3.6 x 10-5 4, 171.0 64.1 16.8 -175.6 150.8 -40.0 -174.2 4.30 -11.0 4.64 -12.7 2.16 3.11 160.3 2.87 3.05 90.7 4.10 4.61 114.9 8.4 x 10-5 2.7 x 10-5 0.949 9 15 8.64 1 4 d l %Bl 176.2 60.2 33.0 -177.5 -173.6 169.8 177.5 7.03 61.6 7.42 57.8 6.42 6.06 -64.5 3.18 3.19 5.1 1 5.16 -81.5 -87.1 3.6 x 10-5 1.1 x 10-5 0.954 609 5.69 26 72 Torsion angles ( w. 6, $) in degrees, distances in angstroms, forces in au, energy (E) in hartrees, and energy differences (AE) in kcal/mol relative to E(yLyL) [-656.963 681 hartree)]. Initial backbone confor- mation (calculated by ECEPP/2). Converged backbone conformation. Cp and CP,, in accordance with classical &turn definition must be shorter than 7 A. In For-Ala-Ala-NH2 the two Ca atoms are replaced by hydrogens (H1 and H4*) (cf. Chart 11); therefore, the model distance (dmdeJ is shorter than CP - CP,, (Figure 1) by no more than 1.1 A. In such a case, T is H1-C2a-C3a-H4. e Critical distances for j3-turn assignment (Cp - CP,,) were extrapolated using ab initio calculated bond lengths and bond angles on the basis of the determined N-H and C-H distances. In such a case, T is Cla-C2*-C3*-C4. /Small Data Base (for the list of nonidentical proteins, see ref 13). g Large Data Base (for the list of proteins, see ref 13). twisted hairpin conformations since Vankatachalam.6 We now use an objective measure, recently defined14 and based on the angularity (or twisting) parameter 7, in agreement with the work 4854 J. Am. Chem. SOC., Vol. 115, No. 11, I993 Table VI. Optimized ab Initio SCF (3-21G) Geometries for Type I and Type. 11' ,!?-Turn Conformations of For-L-Ala-L-Ala-NHf Perczel et al. init conv $1 $1 42 $2 a3 dmodei Tmodel &it Tcrit 01-*HN4 01 --N4 01-HNbN4 01*.*HN3 0 1 *-N3 0 1 -HN 3-N 3 02-*HN4 02-*N4 02- HNbN4 WI W2 max force rms force E AE SDB LDB &&, a L a L , a L h -171.7 -68.6 -17.5 177.8 21.3 176.0 4.83 41.1 5.32 43.7 2.1 1 3.07 162.5 3.01 3.15 3.50 3.98 111.9 5.8 X 10" 0.958 677 3.13 129 435 -113.1 -89.0 1.5 x 10-5 && ~ L c L , -175.9 -121.3 17.6 176.4 169.6 178.1 7.25 19.5 7.56 20.8 7.55 6.98 -51.9 3.63 4.12 112.7 5.10 5.15 -169.4 -87.31 5.6 x 10-5 1.6 x 10-5 0.956 57 4.46 14 37 &&, 6 L 6 D -172.8 -126.8 23.8 171.1 -45.6 -176.5 5.66 -9.9 5.95 -5.6 5.49 5.74 99.4 3.55 4.15 121.2 4.55 4.85 101.8 -173.7 8.7 x 10-5 2.7 x 10-5 0.945 712 11.28 4 9 c D L c D ~ L cD& -177.4 56.1 177.2 26.6 176.5 5.29 5.82 -36.9 1.99 2.98 169.9 3.33 3.21 -74.6 3.34 3.89 115.9 1.4 X 10-4 0.955 958 4.85 1 1 -129.7 -1 12.4 -35.0 4.9 x 10-5 CDYL CDYL -171.9 64.0 -172.8 -175.3 -86.0 66.2 -176.2 5.99 -36.7 6.52 -41.5 3.58 4.19 121.6 4.46 4.09 -62.1 2.05 2.90 141.9 1.3 X lo4 0.952 062 7.29 1 8 4.6 x 10-5 ~ D Y D CDYD -171.3 -178.1 66.9 174.9 75.8 -86.0 -177.7 6.97 71.3 7.52 69.5 5.04 5.91 148.6 4.64 4.23 -59.9 1.91 2.80 146.5 1.8 X lo4 0.947 257 10.31 1 8 3.9 x 10-5 W D C D ~ D -169.6 -178.2 -166.1 -170.6 -179.0 67.7 63.7 8.00 77.8 8.56 75.4 7.79 7.14 -71.7 4.72 4.28 4.61 4.02 -62.1 8.0 X 10-6 2.8 X 10-6 0.940 468 14.57 1 1 -58.2 Units, abbreviations, and parameters are the same as those used in Table V. Scheme I type I ' 8-turns 'DID ' D ' D 'D~D " D ' D INITIAL CEOIIFIRIES FINAL CEOM!ZTFlIES --------> FI type XI' 8-turns %' L INITIAL GEOM!ZTFlIES FINAL GEOMETRIES of Levitt,g* such that for a reverse &turn the value of this T (torsional angle involving Cq, C>l, C>2, C$3 peptide backbone atoms) must be in the -90" 5 T 5 90" range. The following problems were studied: (1) How many of the 81 legitimateI3 backbone conformations of a triamide (e.g., For-Ala-Ala-NH2) would qualify as 8-turns? (2) Can ab initio molecular orbital (MO) computations indicate whether a @-turn has intrinsic stability? If yes, which of the total of 8 1 legitimate minima have such an "intrinsic" stability? (3) Is a 1 - 4-type intramolecular H-bond a necessary structural condition for 8-turns? If not, which types of &turns have a 1 .- 4 hydrogen bond and which do not have such a bond? (4) Can any of the "intrinsically unstable" cy, and/or eL diamide conformations be stabilized and therefore included in a &turn? Results and Discussions In the first part of this study type I' and type 11' @-turn conformations were analyzed ((YDcYD, (YDYD, These conformations were expected to be minimal energy geometries on the basis of previous conformational analyses of ab initio surfaces.12 The computed results are given in Tables V and VI (cf. Scheme I). All three relaxed conformations containing an cyD-typegeometry (LYDCYD, LYDYD, a&) at the first position are typical type I' 8-turns, althoughonly thecyDcyD structureincorporatesa 1 -4-typeH-bond (Table V and Figures 6 and 7). The conformational parameters of eDy, did not change qualitatively during the optimization, and the structure remained a @-turn. Only the optimization of the eDa,-type initial geometry resulted in a qualitatively new backbone (DaL, cDYL, Scheme II pzq --------> 'L 'L ~ ' L ~L ' L' L INITIAL GEOMETRIES FINAL GEOIIFIRIES conformation (eDSL) which is a more "open" conformation (Scheme I). The original t D a L conformation with ~ , D . L =6.47 A and T,D@L =-55.3" values fulfills both the distance (d I 7 A) and the angularity (-90" I T I 90") criteria of a &turn, as does the eDbL geometry ( d r ~ d ~ =5.82 A and T, D~ L =-36.9"). On the basis of an earlier molecular mechanics (MM) study on dipeptide diamides,13 two more conformations (cDyD and cDcD) among the type I'and type 11'8-turns were also incorporated in the analysis. Although the d values of the e D y D and eDeD conformations are longer than expected for a conventional @-turn ( d t D7 D =7.52 A and d,D,D =8.56 A), on the basis of the angularity of the backbone conformation (T value) both of these are hairpin conformations, while the T r ~ + =69.5" and T,D& =75.4" values are smaller than 90" but larger than -90", respectively. In contrast to the above @-turns, the type I @-turns (CYLOLL~ OLLYL, CYL ~L ) as well as the type I1@-turn conformations (CLCYD, BLYD, e L b D ) have an intrinsically unstable aL or eL conformation as their first residue. Starting with the ab initio geometry optimization for the type I 8-turn conformation, using geometries obtained from molecular mechanics, neither the aLaL nor the aLy, conformations were found to beminimum energy structures (Scheme I1 and Table VI). The aLSL structure incorporates a stabilizing intramolecular H-bond, which may suggest that all aL conformations must be stabilized with a hydrogen bond as found in the aLbL conformation. On the basis of the topological analysis of an idealized PES2J 3 or a MM calculation associated with a diamide system, three different type I @-turn structures ((YLcYL, CYLYL, L Y L ~ L ) are expected. These three different backbone conformations were also confirmed by the backbone analyses of X-ray determined protein structures (Table VII); 11 13 Occurrences for the aLcyL, 14 occurrences for the cyL?,, and 129 occurrences for the ( ~ ~ 6 ~ substructures were found in the Small Data Base." The present finding that only the aLbL conformation is a minimum energy structure is congruent with previous ab initio calculations, as this is the only (YLxL or CYLXD J. Am. Chem. SOC.. Vol. 115, No. 11, 1993 4855 -IZOJ I -180 I , , I , 4 5 6 ; I -9- 10 l l C R I T I C AL DIST. ( A) -180 ~ , I , I 1 180 4 ' 4 5 6 7 8 Ib Ill Figure 6. (a) Critical distance (d,,,,) vs T (the angularity of the conformation) of the 75(81) Ac-Ala-Ala-NHCH, conformations, cal- culated by the ECEPP/2 method. (The six conformations marked by *, shown among the 81 symbols in this figure caption below, are the annihilated conformations [for details, see refs 13 and 141) represented by 0 on the top portion of this figure. Conformations incorporating 1 - 4-type H-bonds are plotted as A. The numbers and the conformations they represent are as follows: 1, 5, a D6 L ; C R I T I C AL DI S T ( A ) 2, a D a L ; 3, adB,; 4, 6, ai ~i ; 7, 8, ~ D Y D ; 9, ~ D Y L ; 10, a L a D ; 11, a L a L ; 12, aL @L ; 13, aL 6D; 14, ai6i; 15, ai~i); 16, ~ I Y , ; 17, ~ L c L * ; 18, ~ L Y D ; 19, B L ~ D; 20, B L ~ L ; 21, 6oaI; 30, 6di ; 31,6&; 3276dL; 33,6D~D; 34, 35, ~ D Y D ; 36, ~ Y L ; 37, 61~~1,; 38, 6iai; 39, 6i h; 40, 616,; 41, 42, hie,, 43, 6ifu 44, ~ L Y D ; 45, CI,YII; 54, %YI; 55, Cia,; 56, CL ~L * ; 57, Y L ~ L ; 58, CL@L; 599 e~6D.i 60, L~L*; 61, 1 CD*; 62 ~l ~l ; 63, c I YD; 64, cLYL; 65, Y D ~ D ; 66, YDG 67, YDBL; 689 Y D ~ ; 69, Y I J ; 709 Y n G 71, ~ ~ 6 1 ; 72, YDYD; 73, YDYL; 74, Y L ~ D ; 75, YLBL; 76, Y L ~ D ; 77, ~ ~ 6 , ; 78, yIt D, 79, yIeL; 80, ~ ~ 7 ~ ; 81, -yLyL. (b) d,,,, vs T distribution of the 30 hairpin conformations of For-Ala-Ala-NH2, calculated by the ab initio method. Note that according to the original definition, based on the critical distance9 only 18 of the 30 conformations are @-turns and only 5 (aI,aI, [type HI'@-turn], a D 6 D [type 1'0-turn], aL 6L [type I &turn], c1,6, [type 11' @-turn], and ~ ~ 6 , ) have the 1 - 4-type H-bond (tus). &@I; 22, @I&); 23, 8161; 24, BIG 25, B L t L ; 26, BLYD; 27, B L Y G 28, 29, 6171; 46, Cl)a,; 47, Co al ; 48, CD@L; 49, C D ~ D ; 50, C D ~ L ; 51, CDCD; 52, CDt L*; 53, conformation of For-Ala-Ala-NHz which has an intramolecular H-bond (XL stands for a ~ , &, y ~ , b ~ , and EL; XD stands for CYD, y ~ , 6 ~ , and e ~ ) . The present ab initio calculation resulted in the aLbL (type I &turn) conformation =-69', $,+I =-18', 4 +2 =-113,$1+~=2l0),whichisremarkab1ycloseto thatpredicted by Vankatachalam =-60, $ i + ~ =-30, &+z =-go', tJ1+2 =0'): On the basis of molecular mechanics cal c~l ati ons,~~J ~ three additional 8-turn conformations (adL, aL 6D, and OILEL), each containing the aL subconformation, may also be minimal energy ~tructures.~3,~~ The aLBL conformation was recently assigned to be a &turn by Wilmot and Thortonll on the basis of protein X-ray data analyses, where the torsional angles are close to #,+I =-60, =-30, q$+2 =-120, $,+2 =120'. The present ab initio calculations do not confirm the existence of such an a$, or aLeL conformation for For-Ala-Ala-NH2. These two initial conformers were shifted to 6dL, (Table VI). Although this is a qualitatively new backbone conformation (&fL =-121 O, $:ifL =18', &fL =-169O, #f)tL =170), it has never been Table VII. Frequency of Type I and I' as Well as Type I1 and 11' @-Turn Structures in Selected Proteins" Type I a L a L b ~ L Y L a L & 1113 (8103) 14 (62) 129 (435) Type I' Type 11' 15 (43) 1(8) 1(1) "Frequency values are for the Small Data Base (SDB) and in parentheses for the Large Data Base (LDB) (see ref 13). aLaL triamides may participate in the a-helix, the 3,o helix, or the type I @-turn. Scheme III Fi --------' INIIIAI. GEOMETRIES FINAL GEOMETRIES assigned previously as a 8-turn. It fulfills the angularity criteria of the hairpin conformation with T =20.8'. A similar shift of the aL6, was also observed to bea 6,6, &turn conformation. This second structure seems to be a "perfect" @-turn on the basis both criteria, with a critical distance significantly shorter than 7 A (d =5.95 A) and a backbone angularity rather close to 0 (T =-So). Due to its relatively high energy compared to the L y L confor- mation (AI3 =11.28 kcal/mol), only sporadic Occurrence is expected, as confirmed by X-ray analyses (Table VI). The appearance of 4 (9) representatives of 6,6, in the Small Data Base (SDB) and Large Data Base (LDB)l3confirms the instability of the calculated conformation. Since it is unreasonable to expect that any type of statistical analysis based on protein X-ray data will reveal the existence of such a rare &turn, the present theoretical results are of unique value. According to Table I, all three forms of the type I1 @-turn backbone conformation incorporate an eL substructures in their i+l positions. In all optimizations the eL conformation of the first residue has been shifted to the more stable 6, conformation (Scheme 111). In contrast to preliminary molecular mechanics investigations,13J4 none of these three eLxD conformations were found to be minima for For-Ala-Ala-NH2 according to our ab initio calculations. All three type I1 ,%turn (t ,~,) structures were shifted to the corresponding Sg, minima on the 4D-Ramachandran type map (Scheme 111). Two of the three new backbone conformations and 6DyD) aredistorted and becomeextended conformations. Such a backbone angularity change is authentically monitored14 by the shift AT(~,cY, - aDaD) =-152' - -18' =134" and AT- (cLYD + 6 D y D ) =-163' + -66' =+97'. Although the third conformational switch (~~6, --c 6,6,) resulted in a new backbone category, it still may be regarded as a 8-turn conformation (daD6D =6.87 A and T6D6D =-51.4'). Of the three most important B-turns, type I ( a L a L , ~ L Y L , aL U type 11(eLaD, ~ L Y D ~ and type I11(aLaL), only one (aLaL) was found to be a minimal energy conformation in the ab initio calculations. Using only the X-ray determined 8-turn geometries as input conformations for ab initio studies, even with their mirror images [type I'(aDaO, ~ D Y D , CY&, type 11' ( c D~ L , cDYL, ED&), and type 111' (aDaD) @-turns], no more than seven 8-turn-like backbone conformations can becalculated for For-Ala-Ala-NHz (aDaD, ct,~,, aDbD, eDyL, t D y D , eDe,, and (~~6,). T U Wr. Ab Initio SCF (3-21G) FTum Conformations of For-L-Ala-L-Ala-NH2 Predicted by Multidimensional conformntional Annlysce. init BLW WOV BLUD 0 1 179.2 41 -167.6 $1 168.4 01 172.1 h 62.1 h 35.3 0 3 179.1 d d 7.13 T r y 49.2 dail 7.56 Tai l 49.0 01-HN4 5.96 01-N4 6.69 01-HNCN4 134.8 01-NH3 5.06 01-N3 5.15 OI-NH3-N3 -89.7 02-NH4 3.20 02-N4 3.24 02-NH4-N4 -83.0 BL~D BL- 178.0 -169.0 172.4 -171.5 65.7 -175.5 -179.2 8.43 40.2 8.83 42.8- 8.81 8.17 -47.3 5.05 5.18 91.6 4.55 4.14 -59.8 BLrO BLrO 179.2 -167.2 168.4 172.1 75.6 -57.2 -178.2 7.27 28.0 7.73 31.1 6.29 6.% 128.8 4.99 5.13 92.7 1.93 2.82 146.7 BLTL BLTL 178.9 -167.7 169.3 -177.5 -85.1 68.2 -178.8 7.67 -83.0 8.22 -83.0 6.53 7.16 125.5 5.08 5.15 -88.8 2.04 2.89 140.7 WL WL 176.6 -176.6 -43.6 -178.5 -167.4 170.2 177.7 7.74 -16.2 8.00 -18.4 8.72 8.20 -55.7 4.57 4.87 102.1 5.09 5.14 -87.2 ab aobD 174.2 178.0 -45.6 178.7 -172.9 -49.7 -173.0 6 d D 6.54 -51.5 6.87 -51.4 6.23 6.65 110.7 4.41 4.84 109.8 4.61 4.87 99.7 b L ' b L 175.2 -174.2 154.1 171.7 179.7 6.36 45.2 6.42 43.3 8.1 1 7.37 -39.0 4.46 4.85 107.7 4.53 4.31 -7 1 .O -55.0 -79.0 aDa, awL b 7 L 174.7 178.9 -44.3 -173.2 -85.5 68.8 -179.3 6.89 85.2 7.23 82.2 6.63 7.01 108.8 4.45 4.85 108.0 2.04 2.88 140.3 bD' OLcD -177.0 -161.8 55.7 -152.1 62.6 -173.8 -179.1 6.47 -62.3 6.60 -59.8 8.05 7.33 -40.7 4.01 4.57 I 18.8 4.56 4.18 -61.7 d L d L 175.0 76.1 -5 1.2 -178.7 -161.6 169.2 177.6 7.06 -0.5 7.51 -4.7 6.23 5.54 42. 5 2.01 2.88 144.3 4.95 5.04 -89.2 r D b WD 174.1 73.8 -57.6 178.9 -170.5 4 5 . 4 -179.3 5.81 46. 7 6.34 -47.3 3.19 3.62 107.7 1 .88 2.81 151.8 4.62 4.89 99.7 roak rDh roaL 172.0 75.5 -52.7 -171.9 -122.1 22.9 176.9 5.34 40.4 5.77 38.3 3.61 4.20 120.1 1.91 2.83 149.5 3.54 4.06 114.5 W L YDfL -177.2 72.6 46. 7 160.6 -73.7 168.8 179.0 5.81 57.0 6.09 53.2 5.99 5.34 -45.9 1.90 2.80 147.8 4.35 4.12 -70.1 YWL */DyL 174.3 73.8 -58.3 -173.7 -83.6 67.1 -179.4 6.33 84.4 6.80 80.6 5.26 5.28 -85.3 1.90 2.81 149.1 1.99 2.85 142.3 YLao -172.4 -85.8 64.0 176.6 62.4 33.0 178.9 5.50 -65.5 5.97 -64.9 3.76 4.64 148.5 2.01 2.88 143.6 3.21 3.27 -84.7 nb -177.5 -79.3 75.8 -173.6 176.7 -35.2 -178.2 5.28 43.7 5.84 47.0 2.20 3.14 -157.4 2.07 2.82 130.4 4.58 4.89 102.0 ' WD n- 176.5 -80.9 -1 56.8 64.3 -176.5 -178.9 5.54 -51.2 5.70 -47.9 5.90 5.27 -47.1 1.99 142.8 4.62 4.22 -60.2 75.8 2.85 r& - d L -176.1 -86.8 71.4 -179.7 -164.0 168.6 177.8 7.66 77.7 8.25 78.4 6.40 5.74 -45.0 2.16 2.96 135.4 4.99 5.07 -88.9 nm nn -174.3 -174.4 -84.5 -84.2 68.6 67.0 72.7 -84.9 176.6 -174.1 -51.3 66.4 -177.6 -1789 6.27 7.92 413.5 167.8 6.74 8.76 -79.9 168.4 5.22 5.57 5.33 5.71 90.7 93.1 1.98 2.02 146.6 142.4 1.90 2.00 146.9 142.6 2.87 2.88 2.79 2.86 maxfora ma, force E AE 6.91 8.87 4.34 1.65 7.47 14.33 5.88 7.00 6.11 6.87 8.73 6.11 6.57 2.11 5.09 4.53 5.40 4.17 2.34 0.00 1.5 X 104 2.1 X 104 1.7 X 104 LOX IW 4.7 X IW5 3.1 X IO-' 1.3 X I@' 1.2X 104 1.1 X 1 0 4 1.1 X lW 1.6X 104 6.7 X le5 1.6X 104 6.0X IW5 1.2X 104 1.2X 104 1.7X lW 3.3X 10-5 1.6X l(r 6AX l(r 3.6 X 10-5 4.5 X 10-5 4.4 X 10-5 2.7 X 10-5 1.5 X 10-5 9.7 X I&' 3.8 X 10-5 3.3 X 10-5 2.9 X 1W5 3.0 X IPS 3.6 X 1 V 2.6 X 4.0 X les 3.1 X IO-' 5.1 X 10-5 1.0 X 10-5 4.0 X l(r 2.1 X 105 0.952 663 0.949 539 0.956 752 0.%1 052 0.951 769 0.940 844 0.954 315 0.952 531 0.954 077 0.952 732 0.949 771 0.953 949 0.953 218 0.960 326 0.955 569 0.956 465 0.955 072 0.957 037 0.959 946 0.963 681 4.7 X IC5 1.9 X SDB 12 9 1 20 3 4 9 2 1 1 1 1 2 0 9 2 1 38 9 a 41 113 19 LDB 35 31 3 83 I 1 29 12 5 3 5 3 6 13 5 19 16 5 =163.1O. CThe &*D conformation contaihpan 8-membrrad intramolecular H-bond, where 03-H2 =2.07 A and 03-N2 =2.99 A withan H-bond angle(03-H2-N2) =151.2'. Units, abbreviations. and parameters arc the same as those used inTableV. ' The ~ DCL conformation contains an 8-membered intramolecular H-bond, whcre 03-H2 =2.07 h, 03-N2 =3.05 A with an H-boad angle (03-H2-N2) fl-Turn Conformations Confirmed by Protein X-ray Data J. Am. Chem. Soc., Vol. 115, No. 11, 1993 4851 b ead b r e d b l g d b l g t d l b e d l d d Figure 7. Continued 4858 J. Am. Chem. Soc.. Vol. 115, No. 11, 1993 Perczel et al. Figure7. All 30 &turn conformations resulting from ab initio calculations. By contrast, MDCA considerations predict a significantly larger number of @-turn geometries. Using molecular mechanics (ECEPP/Z), 26 @-turn conformations are expected if the stricter d =7 A criterion is accepted, and a total of 36 different backbone orientations are predicted if the -90' I T 5 90' threshold value is applied. These preliminary considerations were extremely useful, while abinitiocalculations resulted in 18 @-turns according to the stronger distance criteria and an additional 12 (total of 30) (Figure 6b) according to the backbone angularity criteria (-90' <7 <90'). Selected conformational parameters for the 30 @-turn backbone conformations are listed in Tables V, VI, and VIII. The yLaD conformation has a folding pattern similar to that of aL6,, but the middle amide plane is twisted by - 150' (Figure 7). The critical distance is somewhat larger in y L a D (d =5.97 A) than that found in aLgL (d =5.32 A), but the similar absolute value of 7 in both conformations ( T ~L ~L =44', T-,L~D =-65' ) reflects a highly similar degree of folding. This conformation is relatively close to a type 11@-turn. If @-turns are evaluated on the basis of their degree of refolding (T =0), then the y D@L conformation will be the most perfect one ( T ~D ~L =-4.7') (Figure 6b). Such a folding pattern (+,+I 80, J/i+l =d o o , 4i +2 = -160, J/i+2 =160), assigned and labeled here as a @-turn for the first time, was also found in globular proteins (see Table VIII), despite its small probability. conformations (Table VIII) are at least 1 A shorter than the previously defined 7 A as an upper limit for @-turns. On the basis of the backbone angularity value ( 7) , these conformations are perfect hairpin geometries (Figure 7). By contrast, the 1 - 4 intramolecular H-bond is missing in all of these conformations, strongly suggesting that for 8-turn-like geometries such an interaction (1 - 4 H-bond) is not a necessary condition. It seems that such an intramolecular H-bond is necessary where the 8-turn structure incorporates the a, conformational subunit. It is also interesting to note that two different @-turn conformations, 6,y, and yDyL (Table VIII), result in very similar dcrit values as well as almost identical T values, daD,L =7.23 A, dyDtL =6.80 A and 76DyL 82', TyDyL =81, respectively. This suggests that more than a single combination of the subconformations may result in the same degree of hairpin twisting. Up to the present, ab initio calculations have shown that, due to the unfavorable eclipsed interaction of the amide proton and the@ carbon atoms (H-N-Cu-CB torsional angle is approximately -20), the cL backbone conformation is unstable. (As published The critical distances in the a, &, , ?,eD, yLaD, yD6,, and previously,lzJ9-24 the cL backbone conformation was annihilated in For-Gly-NHz, For-Ala-NHZ, and For-Val-NHz.) The &EL and yDc, relaxed conformations (rms forces 3.8 X 10-5 and 4.7 X au, respectively) reported herein are unique exceptions (see Table VI11 and Figure 7). There is no direct interaction (like an intramolecular hydrogen bond) between the third and the second amide groups of the molecule oriented in the t L conformation. However, it is presumable that indirect effects may influence the 6- polarity of the carbonyl oxygen in the central amide. The increased 6- charge on the oxygen can stabihe the eL conformation of the "second half" of the moiecule. This speculation, however, must beinvestigated by ab initiocalculations using larger basis sets and taking correlation effects into account. Conclusion For the first time, ab initio-type calculations on the multidi- mensional conformational problem of dipeptide diamides resulted in a complete set of relaxed @-turn conformations of For-Ala- Ala-NH2. The geometries are intrinsically stable hairpin con- formations. It was shown that if an a,substructureis incorporated in a @-turn conformation (e.g., a,gL in type I @-turn), a favorable H-bond interaction is required to stabilize such a @-turn. By contrast, for @-turns not containing an a, conformational subunit, the existence of such a 1 - 4 H-bond is not required. Thus, while the aDaD conformation contains a 1 - 4-type H-bond, the 6L& folding pattern contains no H-bond3 at all. Therefore, ab initio calculations confirmed that, while the 1 - 4 H-bond may be present in @-turns, it is not a necessary condition for the stabilization of such structures. These calculations lead to the conclusion that the 1 - 4-type intramolecular H-bond is more related to the a,-type substructure than to the true nature of a &turn backboneconformation. The ab initiocalculations reported in this paper resulted in 18 @-turns according to the stronger distance criteria (d I 7 A) and an additional 12 structures (a total of 30 @-turns) according to the backbone angularity criteria Acknowledgment. The authors wish to express their gratitude to the Ontario Center for Large Scale Computing (OCLSC) for the generous allocation of Cray X-MP/28 Supercomputer time. The continued financial support of the NSERC of Canada is gratefully acknowledged. This research was also supported in part by a grant from the Hungarian Scientific Research Foundation (OTKA No. 111-2245). (-90' <7 <90').