The structure and stability of the 16-amino-acid-residue fragment [IG(46–61)] corresponding to the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus was investigated by means of CD and NMR spectroscopy and by differential scanning calorimetry. The CD and 2D NMR experiments were carried out (i) in water at different temperatures and (ii) at one temperature (305 K), with only CD, at different TFE concentrations. Our results show that the IG(46–61) peptide possesses organized three-dimensional structure at all investigated temperatures. The three-dimensional structure of the IG(46–61) peptide resembles the general shape of a β-hairpin that is also observed for this peptide in the experimental structure of the B3 domain in the whole G protein; the structure is stabilized by hydrophobic interactions between nonpolar side chains. Our study shows that the melting temperature of the IG(46–61) peptide is about 320 K which supports the hypothesis that the investigated peptide can serve as a folding initiation site of the B3 domain of the immunoglobulin binding protein G.

We previously studied a 16-amino acid-residue fragment of the C-terminal β-hairpin (residues 46-61), ], of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG(46-61) by systematically shortening the peptide by one residue at a time from both the C and the N terminus. To determine the structure and stability of two resulting 12-and 14-amino acid residue peptides, IG(48-59) and IG(47-60), respectively, we carried out CD, NMR and calorimetric studies of these peptides in pure water. Our results show that IG(48-59) possesses organized three-dimensional structure stabilized by hydrophobic interactions (Tyr50 -Phe57 and Trp48 -Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T m = 310 K) measured by Differential Scanning Calorimetry (DSC). The melting temperature of IG(47-60) determined by DSC is T m = 330 K and its structure is similar to that of the native β-hairpin at all (lower) temperatures examined (283 -313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a β-hairpin structural element.

Two peptides, corresponding to the turn region of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, consisting of residues 51-56 ] and 50-57 ], respectively, were studied by CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β-turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283-323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14 and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β-hairpin structure of peptides corresponding to the C-terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long-range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β-hairpin structure.