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Yingwei Chen, Huaying Zhao, Peter Schuck, Graeme Wistow; Structural Properties of High-methionine Content Fish -crystallins. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2274.
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Fish γ-crystallins accumulate to very high concentrations in lens cells and typically have extremely high methionine content but reduced tryptophan content compared with mammalian γ-crystallins. We examined the consequences of these differences for the structural properties of zebrafish (Danio rerio) γM2a (24 Met, 1 Trp) and γM7-crystallin (17 Met, 2 Trp).
Proteins were expressed in E.coli. Thermal stability and interaction with dimeric human αB-crystallin chaperone were examined by optical density with increasing temperature. Unfolding in guanidinium hydrochloride (GdnHCl) was monitored by circular dichroism (CD) and fluorescence emission. Hydrodynamic properties were investigated by analytical ultracentrifugation (AUC).
The purified proteins had a fluorescence emission peaks between 315-325nm and peak absorbance between 280-285nm. Their far UV CD spectra were typical of the γ-crystallin family, corresponding to a predominantly β-sheet secondary structure. γM2a was stable in solution only up to 15mg/ml and at 0.5mg/ml began to aggregate at 45°C. γM7 could achieve a concentration of at least 40mg/ml and aggregated at 50°C. For both proteins, aggregation was inhibited by αB-crystallin. Increasing concentrations of GdnHCl produced a loss of organized secondary structure, as measured by far UV CD signal at 218nm, for both proteins, with total unfolding at 3.0 M of the denaturant. γM7, with a pair of Trp residues in one domain, showed quenching of Trp fluorescence, as in mammalian γ-crystallins. In contrast, γM2a, with an unpaired Trp showed enhanced fluorescence. By AUC, both proteins exhibited unusual behavior in solution, consistent with a very compact structure, possibly due to low level of hydration.
As isolated proteins, zebrafish γM2a and γM7-crystallins have somewhat lower thermal stability than typical mammalian γ-crystallins. Thus the high methionine content does not seem to contribute to enhanced stability. Neither does it contribute to increased ability to accumulate to very high concentrations, since γM2a has relatively low solubility. However, both proteins exhibit solution properties consistent with a very compact structure that may contribute to the dense packing of total crystallin content in the lens fiber cells. The results from these natural variants also confirm that pairing of Trp residues is required to quench UV fluorescence.
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