NMR evidence for forming highly populated helical conformations in the partially folded hNck2 SH3 domain.
Recent studies of several proteins implied that the folding of beta-proteins may follow a nonhierarchical mechanism in which two major transitions are essential, i.e., the collapse of a random coil to form a nonnative helical intermediate, followed by a transformation into the native beta-structure. We report that the first hNck2 SH3 domain, assuming an all-beta barrel in the native form, can be reversibly transformed into a stable and nonnative helical state by acid-unfolding. We also conducted extensive NMR and mutagenesis studies that led to two striking findings: 1), NMR analysis reveals that in the helical state formed at pH 2.0, the first and last beta-strands in the native form become unstructured, whereas the rest is surprisingly converted into two highly populated helices with a significantly limited backbone motion; and 2), a conserved four-residue sequence is identified on the second beta-strand, a mutation of which suddenly renders the SH3 domain into a helical state even at pH 6.5, with NMR conformational and dynamic properties highly similar to those of the wild-type at pH 2.0. This observation implies that the region might contribute key interactions to disrupt the helical state, and to facilitate a further transformation into the native SH3 fold in the second transition.
Liu
Jingxian
J
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore.
Song
Jianxing
J
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000595:Amino Acid Sequence; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D017510:Protein Folding; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C485544:NCK2 protein, human; D015513:Oncogene Proteins