No abstract

The folding and unfolding reactions of the SH3 domain of spectrin can be described by a two-state model. This domain is a beta-sheet barrel containing 62 amino acids. Equilibrium unfolding by urea, guanidine hydrochloride, and heat is completely reversible at pH values below 4.0. At higher pH values the unfolding is reversible as long as the protein concentration is below 1 mg/mL. The Gibbs energy of unfolding in the absence of denaturant, delta GH2O, at pH 3.5 and 298 K is calculated to be 12 kJ mol-1 for urea, chemical, and temperature denaturation. The stability of the protein does not change noticeably between pH 5.0 and 7.0 and is around 15.5 kJ mol-1. Since heat effects of unfolding are relatively small and, as a result, heat-induced melting occurs in a wide temperature range, the analysis of scanning calorimetry data was performed taking into account the temperature dependence of unfolding delta Cp. The free energy of unfolding obtained for this domain (delta GH2O = 14 +/- 2 kJ mol-1) was, within experimental error, similar to those obtained in this work by other techniques and with those reported in the literature for small globular proteins. Kinetics of unfolding and refolding at pH 3.5, followed both by fluorescence and by circular dichroism, provide evidence of the simplest folding mechanism consistent with the two-state approximation. A value for delta GH2O = 13 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structures of two circular permutants of the alpha-spectrin SH3 domain with new termini within the RT loop (S19-P20s) and the distal loop (N47-D48s) have been determined at 2.02 and 1.77 A resolution respectively. Both fold into the same three-dimensional structure as the wild-type SH3 domain except for the engineered loop that fuses the wild-type termini. The cleaved RT loop in S19-P20s loses nine conserved hydrogen bonds through local hydrogen bond unzipping; no hydrogen bond unzipping occurs in N47-D48s. The structures of the transition states for folding of wild-type alpha-spectrin SH3 domain and the two circular permutants have been examined by analysis of the folding kinetics of eight strategically distributed point mutants. Unlike the native structures, the transition states of the three proteins are considerably different, suggesting that there is no direct relationship between these two states in a protein.

The interaction of six different proline-rich peptides with five SH3 domains has been investigated by using spectroscopic techniques. These peptides correspond to natural sequences and have been implicated in the interaction of some SH3 domains with other proteins. We have determined the Kd values for all of the possible combinations between the peptides and the SH3 domains. Low specificity and low affinity (> 5 microM) are the most remarkable conclusions from these studies. None of the peptides tested here were found to bind with significant affinity to spectrin-SH3 or n-src-SH3. Abl-SH3 seems to be the most selective of the domains analyzed here, while Fyn-SH3 is the most promiscuous. CD and FTIR studies indicate that these peptides adopt to different extents a PPII-like structure in aqueous solution. However, analysis of the SH3 domain complexes with these peptides suggests that proline-rich peptides do not necessarily adopt an overall PPII structure over their entire length upon binding to the different SH3 domains.

The side-chain-side-chain interaction between Phe residues and sulfur-containing residues (Cis and Met) in the two possible orientations at positions i, i + 4 of alpha-helices is described. We have analyzed the contribution to helical stability of the above interactions by studying eight polyalanine-based peptides differing at the residues at positions 9 and 13. These two positions were independently mutated from Ala (AA), to Cys (AC and CA), Met (AM and MA), and Phe (AF and FA) and to the pairs Phe-Met (FM), Met-Phe (MF), Phe-Cys (FC), and Cys-Phe (CF). The intrinsic helical propensities of Cys, Met, and Phe were found to be those previously described in the algorithm AGADIR. NMR analysis of the FM, MF, FC, and CF peptides showed the formation in aqueous solution of contacts between the aromatic ring and the side chains of Cys or Met, at the two i, i + 4 orientations. CD studies demonstrated the important contribution of two of these interactions (FM and FC) to alpha-helix stability (up to 2 kcal mol-1 in the Phe-Cys pair). Statistical analysis of the protein database provides a rationale for the stereospecificity and free energies of the interactions. The very favorable interaction between an aromatic ring and a sulfur-containing amino acid explains why in the protein database around 50% of the sulfur atoms are contacting aromatic rings (Reid et al., 1985).