The excised heat-shock domain of alphaB crystallin is a folded, proteolytically susceptible trimer with significant surface hydrophobicity and a tendency to self-aggregate upon heating

Protein Expression and Purification
Bishwajit KunduPurnananda Guptasarma

Abstract

The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains...Continue Reading

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Citations

Aug 31, 2010·Nature Structural & Molecular Biology·Stefan JehleHartmut Oschkinat
Aug 12, 2010·Bioscience, Biotechnology, and Biochemistry·Tofazzal HossainYoichi Aso
Apr 6, 2011·Proceedings of the National Academy of Sciences of the United States of America·Stefan JehleRachel E Klevit
Jan 4, 2015·Biochimica Et Biophysica Acta·Raman BakthisaranCh Mohan Rao
Aug 16, 2016·Biochimica Et Biophysica Acta·Puttur SanthoshkumarKrishna K Sharma
Nov 7, 2006·IUBMB Life·G Bhanuprakash ReddyM Satish Kumar

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