Synthesis, folding, and structure of the β‐turn mimic modified B1 domain of streptococcal protein G

Abstract

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

Folding thermodynamics of protein-like oligomers with heterogeneous backbones

“…Supporting the validity of the measurements, values obtained for wild-type GB1 ( 1 ) showed good agreement with previously reported data. 18,35,36 …”

Observing the osmophobic effect in action at the single molecule level

“…From this category, one of the smallest model proteins that are extensively used in protein stability studies is the B1 immunoglobulin‐binding domain of protein G from Streptococcus , commonly referred to as GB1. Protein GB1 has been used to study protein‐related topics as diverse as electrostatic and salt‐screening contributions to protein stability,56 mechanisms of beta‐sheet formation,58 biomimetic materials,59, 60 and even protein aggregation 61, 62. The existing extensive characterization of the structure, thermodynamics and kinetics of GB1,54, 63–71 together with its high mechanical stability29, 59 and the fact that it unfolds mechanically without any observable intermediates on AFM time scales make from GB1 a very compelling choice for the study of the osmophobic effect at the single molecule level.…”

Molecular dynamics characterisations of the Trp-cage folding mechanisms: in the absence and presence of water solvents

“…The incorrect bonds of the Trp-cage structure were modified using the Biopolymer module, and then saturated with the hydrogen atoms on the basis of the expected charge distribution at physiological pH [30,31]. The N-and C-termini were capped with the acetyl and methyl groups, respectively [32,33]. The total charge of the resulting system is 1.0 and is neutralised by one chloride anion (Cl 2 ).…”