Insights into protein aggregation by NMR characterization of insoluble SH3 mutants solubilized in salt-free water.
Protein aggregation in vivo has been extensively associated with a large spectrum of human diseases. On the other hand, mechanistic insights into protein aggregation in vitro were incomplete due to the inability in solubilizing insoluble proteins for high-resolution biophysical investigations. However, a new avenue may be opened up by our recent discovery that previously-thought insoluble proteins can in fact be solubilized in salt-free water. Here we use this approach to study the NMR structural and dynamic properties of an insoluble SH3 mutant with a naturally-occurring insertion of Val22 at the tip of the diverging turn. The obtained results reveal: 1) regardless of whether the residue is Val, Ala, Asp or Arg, the insertion will render the first hNck2 SH3 domain to be insoluble in buffers. Nevertheless, all four mutants could be solubilized in salt-free water and appear to be largely unfolded as evident from their CD and NMR HSQC spectra. 2) Comparison of the chemical shift deviations reveals that while in V22-SH3 the second helical region is similarly populated as in the wild-type SH3 at pH 2.0, the first helical region is largely unformed. 3) In V22-SH3, many non-native medium-range NOEs manifest to define non-native helical conformations. In the meanwhile a small group of native-like long-range NOEs still persists, indicating the existence of a rudimentary native-like tertiary topology. 4) Although overall, V22-SH3 has significantly increased backbone motions on the ps-ns time scale, some regions still own restricted backbone motions as revealed by analyzing (15)N relaxation data. Our study not only leads to the establishment of the first high-resolution structural and dynamic picture for an insoluble protein, but also shed more light on the molecular events for the nonhierarchical folding mechanism. Furthermore, a general mechanism is also proposed for in vivo protein aggregation triggered by the genetic mutation and posttranslational modification.
Liu
Jingxian
J
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Song
Jianxing
J
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009038:Motion; D009154:Mutation; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012492:Salts / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D004220:Disulfides; C485544:NCK2 protein, human; D015513:Oncogene Proteins; D011506:Proteins; D012492:Salts