Structure and Assembly of Designed -Hairpin Peptides in Crystals as Models for -Sheet Aggregation †,‡ Subrayashastry Aravinda, § Veldore Vidya Harini, § Narayanaswamy Shamala,* ,§ Chittaranjan Das, | and Padmanabhan Balaram* ,| Department of Physics and Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India ReceiVed August 26, 2003; ReVised Manuscript ReceiVed December 15, 2003 ABSTRACT: De noVo designed -hairpin peptides have generally been recalcitrant to crystallization. The crystal structures of four synthetic peptide -hairpins, Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (2), Boc-Leu-Val-Val-DPro-Aib-Leu-Val-Val-OMe (3), and Boc-Met-Leu-Phe-Val-DPro-Ala-Leu-Val-Val-Phe-OMe (4), are described. The centrally positioned DPro- Xxx segment promotes prime -turn formation, thereby nucleating -hairpin structures. In all four peptides well-defined -hairpins nucleated by central type II′ DPro-Xxx -turns have been characterized by X-ray diffraction, providing a view of eight crystallographically independent hairpins. In peptides 1-3 three intramolecular cross-strand hydrogen bonds stabilized the observed -hairpin, with some fraying of the structures at the termini. In peptide 4, four intramolecular cross-strand hydrogen bonds stabilized the hairpin. Peptides 1-4 reveal common features of packing of -hairpins into crystals. Two-dimensional sheet formation mediated by intermolecular hydrogen bonds formed between antiparallel strands of adjacent molecule is a recurrent theme. The packing of two-dimensional sheets into the crystals is mediated in the third dimension by bridging solvents and interactions of projecting side chains, which are oriented on either face of the sheet. In all cases, solvation of the central DPro-Xxx peptide unit -turn is observed. The hairpins formed in the octapeptides are significantly buckled as compared to the larger hairpin in peptide 4, which is much flatter. The crystal structures provide insights into the possible modes of -sheet packing in regular crystalline arrays, which may provide a starting point for understanding -sandwich and cross--structures in amyloid fibrils. -Sheets are widely distributed in protein structures. In globular proteins, multistranded -sheets are generally buried in protein interiors and are stabilized by networks of interstrand hydrogen bonds (1, 2). In membrane proteins the -barrel, a closed -sheet assembly facilitated by the intrinsic curvature of sheets, is a widespread structural feature (3). Quite often, antiparallel strands in protein structures are connected by tight turns, generally two-residue -turns, with registered cross-strand hydrogen bonds orienting the anti- parallel segments of the polypeptide chain, resulting in hairpin formation (4-8). Considerable recent interest in -sheet structures stems from their occurrence in insoluble polypeptide deposits, which form amyloid-like fibrils (9- 11). Attempts to design -sheets from “first principles” have focused on -hairpins as the fundamental unit in generating multistranded structures (12-15). X-ray diffraction studies of -hairpin peptides in single crystals can provide invaluable information on the packing and three-dimensional assembly of extended polypeptide strands. We have therefore been investigating the design and construction of -hairpin pep- tides with a high propensity to assemble into single crystals. The use of stereochemically constrained amino acids in the design of conformationally well-defined peptides has attracted attention in recent years. Helical folding in designed sequences has been achieved by the incorporation of R-ami- noisobutyric acid (Aib) 1 and related C R,R dialkylated residues (15-17). The construction of -hairpin structures has been approached by the incorporation of centrally located DPro- Xxx segments, which stabilize the formation of prime -turns (type I′/II′), serving as nuclei for the formation of antiparallel -strands registered by interchain hydrogen bonds (18-24). The use of DPro-Xxx segments is a consequence of analysis of -hairpins in protein structures, which revealed that proper registry of antiparallel strands is often realized by nucleating turns favoring type I′/II′ conformations (4-7). In the DPro residue the torsion angle φ is restricted to a value of +60° ( 20°, constraining DPro-Xxx -turns which incorporate this residue at the i + 1 position (15). Recent studies from this laboratory have demonstrated the utility of centrally placed DPro-Xxx segments in nucleating isolated -hairpins (25- 29) and in the generation of multistranded -sheet structures (30-33). Crystal structure determination and detailed NMR † This work was supported by a grant from the Council of Scientific and Industrial Research and a program grant in the area of Molecular Diversity and Design by the Department of Biotechnology, Government of India. V.V.H. is a Senior Research Fellow of the Council of Scientific and Industrial Research, Government of India. ‡ Atomic coordinates have been deposited in the Cambridge Crystal- lographic Data Centre: CCDC 218047, CCDC 218048, CCDC 218049, and CCDC 218050. * To whom correspondence should be addressed. N.S.: fax, 91-80- 23602602/91-80-23600683; e-mail, shamala@physics.iisc.ernet.in. P.B.: fax, 91-80-23600683/91-80-23600535; e-mail, pb@mbu.iisc.ernet.in. § Department of Physics. | Molecular Biophysics Unit. 1 Abbreviations: Aib, R-aminoisobutyric acid; DPro, D-proline. 1832 Biochemistry 2004, 43, 1832-1846 10.1021/bi035522g CCC: $27.50 © 2004 American Chemical Society Published on Web 01/29/2004