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Insights into protein aggregation by NMR characterization of insoluble SH3 mutants solubilized in salt-free water.

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 (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.

SUBMITTER: Liu J

PROVIDER: S-EPMC2776303 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Insights into protein aggregation by NMR characterization of insoluble SH3 mutants solubilized in salt-free water.

Liu Jingxian J Song Jianxing J

PloS one 20091123 11

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 structura ...[more]

PMID: 19956763

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Project description:In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the ?-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media.

Dataset Information

Insights into protein aggregation by NMR characterization of insoluble SH3 mutants solubilized in salt-free water.

Publications

Insights into protein aggregation by NMR characterization of insoluble SH3 mutants solubilized in salt-free water.

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