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J. M. Skiba, A. F. Drossman, A. J. Kiss, J. F. Hejtmancik, Y. Sergeev, M. Posner; Comparison of Thermally Adapted Fish A-Crystallins Identifies an Amino Acid Substitution Associated With Chaperone-Like Activity and Thermal Stability. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4614.
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© ARVO (1962-2015); The Authors (2016-present)
To use bony fish αA-crystallins as a model group to identify amino acid substitutions affecting thermal stability and chaperone-like activity and gain insights into small heat shock protein evolution.
Purified recombinant αA-crystallins from six fish species were previously cloned and used to identify correlations between physiological temperature, protein hydrophobicity, chaperone activity and thermal stability. The TreeSAPP algorithm identified αA-crystallin regions undergoing natural selection for changes in hydrophobicity. Homology modeling using 3D-PSSM and PHYRE servers identified specific amino acid substitutions differing in hydrophobicity that were predicted to affect chaperone-like activity. Modified zebrafish αA-crystallins differing in hydrophobicity at these residues were produced using PCR-based site directed mutagenesis, and the chaperone-like activity of each recombinant protein was quantified by measuring its ability to prevent the chemically induced aggregation of lactalbumin. Thermal stability of each protein was measured by monitoring aggregation at increasing temperatures.
Analysis of molecular adaptation and homology modeling of six bony fish αA-crystallins identified several residues differing in hydrophobicity that were predicted to affect thermal stability and chaperone-like activity. The single substitution V62T in zebrafish αA-crystallin decreased thermal stability and increased chaperone-like activity compared to the wild type zebrafish protein. A second engineered mutation (C144S) did not alter thermal stability or chaperone-like activity at temperatures between 20-35°C, but showed minor elevated protein binding activity at 40°C. Molecular modeling of the zebrafish αA-crystallin domain suggests that the V62T mutation affects the structural stability of β-sheets. The C144S mutation is exposed at the domain surface and is expected to have a lesser effect on αA-crystallin stability.
These data indicate that change in a single amino acid residue can partially account for the thermal adaptation of vertebrate αA-crystallins, and that differences in hydrophobicity may underlie the functional effect of this variation. Furthermore, this study validates a comparative evolutionary approach to analyzing structure/function relationships in small heat shock proteins, a protein family involved in numerous human diseases.
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