The mechanism of b-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied b-turn mimic, designated as the dibenzofuran-based~DBF! amino acid, in the B1 domain of streptococcal protein G~B1G!, and compare our results with those obtained upon insertion of the same mimic into the N-terminal b-hairpin of B1G~O Melnyk et al., 1998, Lett Pept Sci 5:147-150!. The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the b-turn mimic from acting as a strong b-sheet nucleator, which reinforces the idea that the native b-hairpin formation is not driven by the b-turn formation, but by tertiary interactions.Keywords: activity; b-turn mimic; dibenzofuran; domain B1 of protein G; folding; protein engineering; stability; structure Understanding how a protein folds into its native form still remains a question of fundamental interest for the de novo design of proteins. Peptide studies on a-helix derived fragments support the framework model, with secondary structure elements formed during the initiation of the protein folding process~Dyson et Freund et al., 1996!, and To impose a suitable chain reversal and a correct hydrogen bonding pattern of the directly attached amino acids allowing the formation of the first stabilizing hydrogen bond of the b-sheet, a myriad of synthetic b-turn mimics have been designed and evaluated on their capacity to nucleate b-sheets in small peptides~Baca et al., 1993;Kemp & Li, 1995;Nesloney & Kelly, 1996a, 1996b One example is the dibenzofuran-based b-turn mimic~Fig. 1B! designed by Díaz and Kelly~1991! that has been thoroughly characterized and successfully incorporated into several small peptides Díaz et al., 1992Díaz et al., , 1993aDíaz et al., , 1993bTsang et al., 1994!. We recently have set out to incorporate this b-sheet nucleator into a real protein environment as defined by the existence of a full tertiary structure. Scyllatoxin, a 31 amino acid scorpion toxin with Reprint requests to: G. Lippens, Laboratoire Synthèse, Structure, Fonction des Biomolécules CNRS UMR 8525, Institut de Biologie de Lille, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 447 59021 Lille Cedex, France; e-mail: guy.lippens@pasteur-lille.fr.Abbreviations: 1D, 2D, 3D, one-, two-, and three-dimensional; B1G, B1 domain of the streptococcal protein G; B1G-DBF, DBF-modified B1 domain of the streptococcal protein
