Abstract
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 15N 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.
Highlights
Protein aggregation in cells is emerging as common features of the diseases, in particular for a large array of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases
We recently reveal that the first human Nck2 SH3 domain adopting a classic SH3 fold in the native condition [18] suddenly becomes highly helical upon being destabilized by acid at pH 2.0 or 4-residue mutations on the second b-strand [11]
We have discovered that protein insolubility could be overcome by suppressing attractive hydrophobic interactions with intrinsically repulsive electrostatic interactions which are expected to have the largest strength in salt-free water [4,5,6,7,8,9,10,11]
Summary
Protein aggregation in cells is emerging as common features of the diseases, in particular for a large array of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases. Recently we discovered that the previously-thought insoluble proteins, one even with transmembrane fragment, could be solubilized in salt-free water for detailed biophysical studies [4,5,6,7,8,9,10,11]. This approach has been used by other groups to investigate aggregation-prone proteins [12,13,14]
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