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A Biophysical and Structural Investigation of Human (gamma)s-crystallin and the Formation of Cataracts

出版	University of California, Irvine, 2011
ISBN	12670792909781267079299
URL	http://books.google.com.hk/books?id=DR5BnQAACAAJ&hl=&source=gbs_api

註釋A point variant (G18V) of (gamma)S-crystallin, a human eye lens protein, has been documented to cause childhood-onset cataracts in humans. The molecular mechanisms that contribute to cataract formation in general remain unclear, and studies on clinically documented variants that cause cataracts will allow a better understanding of protein aggregation. Evidence in this study indicates that the formation of (gamma)S-G18V-related cataract is likely due to disulfide linkages between newly exposed cysteines or exposure of the hydrophobic V18 sidechain at high protein concentrations. (gamma)S-crystallin was cloned, expressed, and purified from E. coli, along with two aggregation-prone variants, (gamma)S-G18V and (gamma)S-G106V. All three variants were characterized using optical spectroscopy and biophysical methods to compare their aggregation propensities and thermodynamic stabilities. (gamma)S-WT and (gamma)S-G18V were examined using NMR spectroscopy to determine both the suitability of the healthy, soluble, wild-type protein for solid-state NMR measurements, and the atomic-resolution liquid-state NMR structures of the wild-type protein and the G18V variant. Biochemical analysis of all three variants indicated they were fully folded and primarily & beta;-sheet in structure. The biophysical analysis indicated that (gamma)S-G18V and (gamma)S-G106V show a separation of thermodynamic stability and solubility due to sequence-specific differences between the two domains: (gamma)S-G18V is more stable and (gamma)S-G106V is more soluble at neutral pH. Concentrated (gamma)S-WT in a hydrogel-like state is an appropriate sample for solid-state NMR structural studies, and detailed information about its intermolecular structure can be gathered using standard solid-state methods. Liquid-state NMR structures of (gamma)S-WT and (gamma)S-G18V were calculated and show that (gamma)S-G18V undergoes extensive structural disruption in its N-terminal domain as a result of the G18V substitution, but does not expose V18 to the solvent. Instead, (gamma)S-G18V exposes several cysteines that are likely candidates for the mechanism of aggregation. Two additional NMR structures refined using subsets of the (gamma)S-G18V shifts suggest that the observed concentration-dependent aggregation of (gamma)S-G106V may be due to the exposure of the hydrophobic valine sidechain, which does not remain buried in (gamma)S-G106V. A model describing the structural, thermodynamic, and biophysical differences between (gamma)S-WT, (gamma)S-G18V, and (gamma)S-G106V has been proposed that explains the accumulated data on these three proteins.