Collateral Existence of Folded and Extended Conformations of the -Ala Moiety in a Model Peptide Ashwani Kumar Thakur,* Paloth Venugopalan,† and Raghuvansh Kishore* ,1 *Institute of Microbial Technology, Sector 39-A, Chandigarh-160 036, India; and †Department of Chemistry, Panjab University, Sector 14, Chandigarh-160 014, India Received May 29, 2000 The single-crystal X-ray diffraction analysis of a nonchiral -Ala-containing model peptide, Boc-- Ala-Aib-OCH 3 1 (-Ala, 3-aminopropionic acid; Aib, -aminoisobutyric acid), establishes the coexistence of distinctly different backbone conformations in two crystallographically independent molecules, A and B, in the asymmetric unit. Interestingly, the central  torsion angle around the -C  –C  - bond of the confor- mationally flexible -Ala residue appears to be critical in dictating the overall distinct structural features, i.e., in molecule A it adopts a folded gauche conforma- tion:  71.0°, whereas it favors an extended trans conformation,   161.2°, in molecule B. As expected, the stereochemically constrained Aib residue pre- ferred an energetically favorable folded backbone con- formation, the torsion angles being   46.2° and   48.3° for molecule A and  43.6° and  45.5° for molecule B, lying in the left-handed and right-handed helical regions of the Ramachandran map, respec- tively. Considering the signs as well as the magnitudes of the backbone torsional angles, molecule A typically folds into a pseudo type III -turn-like structure while molecule B prefers an overall extended conformation. Entrapping the two dramatically distinct conforma- tional characteristics in the crystalline state clearly suggests that the gauche and the trans effects of the -Ala moieties are indeed energetically accessible to a short linear peptide and receive strong experimental support. The analyses permitted us to emphasize that in addition to conformational constraints of the neigh- boring residue, the chemical nature of the side-chain acyclic substituents and the “local environments” col- lectively seem to influence the stabilization of the folding– unfolding behavior of the two methylene units (-CONH-CH 2 -CH 2 -CONH-) in 1. © 2000 Academic Press Key Words: -Ala peptide; peptide design; X-ray dif- fraction analysis; folded– unfolded -Ala moities. In the past decade there has been increasing interest in de novo design and construction of secondary struc- tural features in short linear peptides incorporating conformationally flexible noncoding -Alanine (1–20). Relatively limited conformational investigations per- formed on short linear -Ala peptides and derivatives reveal that despite its inherent flexibility the residue is capable of accommodating novel well determined three-dimensional structures mimicking the pieces of various secondary structures, frequently observed in proteins and polypeptides. Additionally, several publi- cations have also witnessed the use of this amino acid for a variety of functional investigations since, the flex- ibility not only provides possibility of interaction with different receptors but also reduce their enzymatic bio- degradation significantly (21, 22). The paucity of infor- mation about the conformational adaptability of this flexible residue impeded our ability for rationale pre- dictions of the structure–function relationships of the bioactive peptides incorporated -Ala residue(s) (23– 28). However, an improved understanding of the atomic spatial organization and informations on the topography of the conformational requirements, i.e., incorporation/substitution of the -Ala residue ex- pected to assisted in design and development of the bioactive peptidomimetics analogs exhibiting high po- tency and/or selectivity (29 –33). The analysis of conformational investigations per- formed on -Ala peptides however, revealed that the unsubstituted -amino acid is capable of controlling the overall topographical properties of the peptides. For instance the potentials of this residue in (i) mod- eling the conformational features reminiscent of polyg- lycine II proposed by Crick and Rich (5) (ii) accommo- dating the chirality induced non-superimposable stereogeometrical features (14, 19) and novel helical folds (8 –10) (iii) the formation of folded “-turn like” structural motifs, stabilized by conventional and non- conventional intramolecular interactions, are of special relevance (11, 18). The conformational preferences 1 To whom correspondence should be addressed. Fax: 0091-172- 690585. E-mail: kishore@imtech.ernet.in. Biochemical and Biophysical Research Communications 273, 492– 498 (2000) doi:10.1006/bbrc.2000.2966, available online at http://www.idealibrary.com on 492 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.