9632 J. Am. Chem. zyxwvu SOC. 1995,117, 9632-9637 Peptide Design. Structural Evaluation of Potential Nonhelical Segments Attached to Helical Modules Isabella L. Karle,*J R. Gurunath,’ Sudhanand Prasad,’ Ramesh Kaul,* R. Balaji Rao? and P. Balaram*,’ Contribution from the Laboratory zyxwvut for the Structure zyxwvu of Matter, Naval Research Laboratory, Washington, D. C. 20375-5000, Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India, and Department of Chemistry, Banaras Hindu University, Varanasi-221 005, India Received June 6, 1995@ Abstract: The conformations of three decapeptides containing a helical heptapeptide module attached to a potentially helix destabilizing tripeptide segment have been investigated in single crystals. X-ray diffraction studies of the sequence Boc-Gly-Dpg-Xxx-Val-Ala-Leu-Ab-Val-Ala-Leu-OMe (Xxx zyxwv = Leu (l), Pro (2), and Ala (3); Dpg = a,a-di-n-propylglycine; Aib = a-aminoisobutyric acid) reveal helical conformations for the segment 2-9 in all three peptides. In 1 and 2 Gly(1) is not accommodated in the right-handed helix and adopts a left-handed helical conformation with positive zyxwvuts 4, zyxwvutsr q!~ values. The terminal blocking group extends away from the helix in 1 and 2. In 3 the helix is continuous, encompassing residues 1-9. The Dpg residues in all three cases adopt helical conformations, even when flanked by two helix destabilizing residues as in 2. These findings suggest that the higher a,a-dialkyl residues are good helix promoters although theoretical calculations suggest the existence of a pronounced energy minimum in fully extended regions of conformational space. None of the peptides pack efficiently. The register between helices in the head-to-tail region is not good, with disordered water molecules serving as hydrogen bond bridges and as space fillers. The crystallographic parameters follow. 1: Xxx = Leu, C ~~H~~NIOOI~*~H~~~H~OH P212121, a = 16.399(3) A, b = 18.634(3) A, c = 23.241(4) A. 2: Xxx = Pro, C~~H~~NIOOI~*XH~O, P212121, a = 16.468(4) A, b = 18.071(4) A, c = 23.397(5) A. 3: Xxx = Ala, C~IH~~NIOOI~*XHZO, P21212, a = 19.289(7) A, b = 35.950(12) A, c = 9.570(3) A. Introduction Nonstandard amino acids with strong conformational prefer- ences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design.’ The strong helix inducing properties of a-aminoisobutyric acid (Aib),2 the first member of the series of a,a-di-n-alkyl glycines, have been well documented by numerous crystal structure analyses of short peptides3 that form 310-helicesand longer peptides4 that form mixed 3lo-/a-helices and a-helices. Incorporation of even a single Aib residue at the center of a 7 or 9 residue sequence is sufficient to stabilize a two-to-three tum helical This ability to construct stereochemically well defined helical peptide modules may be used to advantage in a “Meccano (or Lego) Set” approach to the design of super-secondary structure motifs.’ Naval Research Laboratory. * Indian Institute of Science. 0 Banaras Hindu University. @Abstract published in Advance ACS Abstracts, September 1, 1995. (1) (a) Balaram, P. Pure Appl. Chem. 1992,64, 1061-1066. (b) Balaram, P. Curr. chin. Struct. Biol. 1992. 2. 845-851. (c) Karle. I. L.: FlioDen- Anderson, ‘J. L.; Sukumar, M.; Uma, K.; Balaram,‘ P. J. Am. Chem: koc. 1991, 113, 3952-3956. (2) (a) Abbreviations used: Aib, a-aminoisobutyric; Dpg, a,a-di-n- propylglycine; Boc, tert-butyloxycarbonyl. All chiral amino acids are of the L configuration. (b) Conventions used for torsion angles in peptides follow the IUPAC-IUB Commission on Biochemical Nomenclature. Biochemistry 1970, 9, 3471-3479. (3) (a) Toniolo, C.; Bonora, G. M.; Bavoso, A,; Benedetti, E.; DiBlasio, B.; Pavone, V.; Pedone, C. Biopolymers 1983, 22, 205-215. (b) Prasad, B. V. V.; Balaram, P. CRC Crit. Rev. Biochem. 1984, zyxwvutsr 16, 307-347. (4) (a) Karle, I. L.; Balaram, P. Biochemistry 1990 29, 6747-6756. (b) DiBlasio, B.; Pavone, V.; Saviano, M.; Lombardi, A.; Nastri, F.; Pedone, C.; Benedetti, E.; Crisma, M.; Anzolin, M.; Toniolo, C. J. Am. Chem. zyxwvut SOC. 1992,114,6273-6278. (c) Karle, I. L.; Flippen-Anderson, J. L.; Gurunath, R.; Balaram, P. Prorein Sci. 1994, 3, 1547-1555. 0002-7863/95/1517-9632$09.00/0 Construction of a helix-linker-helix motif with a well-defined orientation of the two cylindrical peptide elements could be achieved, in principle, by controlling the stereochemistry of the linking segment. While construction of continuous helical modules encompassing three-to-four helical turns has been achieved, with structures of 13 to 16 residue sequences being characterized in crystal^,^ control over linking sequence con- formations has proved more difficult. Initial attempts to generate peptides with two distinct helical segments have used c-aminocaproic acid (Acp),IC D-residues,6 and Pro7 as intermpt- ing elements. While Acp incorporation results in clearly demarcated helical segments, D-residues and Pro have been accommodated in the framework of right-handed peptide helices in 16-residue sequences. The higher a,a-dialkyl amino acids like a,a-di-n-propylgly- cine (Dpg) have been shown to adopt fully extended (C5,# = 2y = 180’) conformations in crystal structures of short ho- mopeptides.8 In longer heteromeric sequences they are accom- modated in helical structure^.^ Coexistence of both extended and helical conformations of Dpg in crystals of a tripeptide, Boc-Leu-Dpg-Val-OMe, suggests that the two conformations are approximately isoenergetic,I0 with environmental factors being critical in tilting the balance. It was therefore of interest (5) (a) Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Balaram, P. Biochemistry 1989, 28, 6696-6701. (b) Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Sukumar, M.; Balaram, P. J. Am. Chem. SOC. 1990,112,9350- 9356. (c) Karle, I. L.; Flippen-Anderson, J. L.; Sukumar, M.; Balaram, P. J. Med. Chem. 1992, 35, 3885-3889. (6) Gurunath, R.; Balaram, P. Biochem. Biophys. Res. Commun. 1994, (7) Uma, IC.; Karle, I. L.; Balaram, P. In Proteins: Structure, Dynamics and Design; Renugopalakrishnan, V., Carey, P. R., Smith, I. C. P., Huang, S. G., Storer, A., Eds.; ESCOM Science Publishers: B. V. Leiden, 1991; pp 295-301. 202, 241-245. 0 1995 American Chemical Society