Circular dichroic investigation of the native and non-native conformational states of the growth factor receptor-binding protein 2 N-terminal src homology domain 3: Effect of binding to a proline-rich peptide from guanine nucleotide exchange factor

SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain fro m growth factor receptor-binding protein 2 (Grb2), an adapter protein in th e intracellular signaling pathway to Ras, was investigated by circular dich roic (CD) spectroscopy. The compact native beta-barrel conformation, previo usly elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, i n the absence of salt. From the structural changes induced by varying pH, i onic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the f ar- and near-UV domains, Along the free energy scale, these appear to distr ibute into two conformational ensembles, depending on the extent of structu ral and thermodynamic differences compared to the native conformation, The first ensemble consists of non-native conformations with a nativelike secon dary structure, and the second is composed of partially unfolded conformati ons having short alpha-helical fragments or turnlike motifs in their nonnat ive secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactne ss of their inner structure, depending on the strength of nonlocal interact ions, but only the native all-beta conformation possesses a condensed prote in exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary stru cture change in a hydrophobic pocket at the binding site. This is accompani ed by the PP-II helix folding of the proline-rich peptide. interestingly, i n the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of th e binding process.