Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin-binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

Abstract

A 34-residue alpha/beta peptide [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the beta-hairpin in the native structure, forms structure similar to the beta-hairpin only at T = 313 K, and the structure is stabilized by non-native long-range hydrophobic interactions (Val47-Val59). On the other hand, the N-terminal part of IG(28-61), which corresponds to the middle alpha-helix in the native structure, is unstructured at low temperature (283 K) and forms an alpha-helix-like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long-range connectivities which would have supported packing between the C-terminal (beta-hairpin) and the N-terminal (alpha-helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726-736), based on Monte-Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed.

Figure 1

(a) The X-ray structure of the B3 domain of protein G (1IGD). (b) The amino acid sequence of 1IGD. In (b), A denotes β-strands and H1 the α-helix. The boxed part of the sequence, IG(28-61), was studied in this work.

Figure 2

Heat capacity curve for IG(28-61) recorded in water at pH = 6.02.

Figure 3

CD spectra of IG(28-61) in (a) water at 16 different temperatures (pH = 6.02) and (b) water solutions of trifluoroethanol (10%, 50% and 90% of TFE).

Figure 4

ROE effects corresponding to the interproton contacts and the ³J_NHHα coupling constants of IG(28-61) measured in H₂O at (a) 283 K, (b) 305 K and (c) 313 K. The thickness of the bars reflects the strength of the ROE correlation as strong, medium or weak.

Figure 5

The lowest-energy conformation which is the representative conformation from the most populated family of conformations of IG(28-61) obtained by using time-averaged MD methodology with restraints from NMR measurements at 283 K. (a) The front view of the structure, (b) the side view of the structure.

Figure 6

The lowest-energy conformation which is the representative conformation from the most populated family of conformations of IG(28-61) obtained by using time-averaged MD methodology with restraints from NMR measurements at 305 K.

Figure 7

The lowest-energy conformation which is the representative conformation from the most populated family of conformations of IG(28-61) obtained by using time-averaged MD methodology with restraints from NMR measurements at 313 K. (a) The front view of the structure, (b) the side view of the structure.