A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallin
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Date
2010-11-01
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Elsevier
Abstract
The principal lens proteins alpha A- and alpha B-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, alpha A- and alpha B-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. alpha B-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either alpha A- or alpha B-crystallin is not known nor is the mechanism of their chaperone action. Our earlier H-1 NMR spectroscopic studies determined that mammalian sHsps have a highly dynamic, polar and unstructured region at their extreme C-terminus (summarized in Carver (1999) Prog. Ret. Eye Res. 18, 431). This C-terminal extension acts as a solubilizing agent for the relatively hydrophobic protein and the complex it makes with its target proteins during chaperone action. In this study, alpha A- and alpha B-crystallin were N-15-labelled and their H-1-N-15 through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. H-1-N-15 spin-lattice (T-1) and spin spin (T-2) relaxation times were measured for alpha A- and alpha B-crystallin in the absence and presence of a bound target protein, reduced alpha-lactalbumin. H-1-N-15 Nuclear Overhauser Effect (NOE) values provide an accurate measure, on a residue-by-residue basis, of the backbone flexibility of polypeptides. From measurement of these NOE values, it was determined that the flexibility of the extension in alpha A- and alpha B-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of alpha A-crystallin with reduced alpha-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of alpha B-crystallin in its C-terminal region: (i) 1159A and 1161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One 2, e1046). Comparison of H-1-N-15 NOE values for these mutants with wild type alpha B-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L alpha B-crystallin, which may affect chaperone ability. (C) 2010 Elsevier Ltd. All rights reserved.
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Keywords
Heat-shock proteins, Quantitative chemical analysis, NMR spectra, Polypeptides, Crystalline lens
Citation
Treweek, T.M., Rekas, A., Walker, M.J., Carver, J.A. (2010). A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallin, Experimental Eye Research, 91(5), 691-699. 10.1016/j.exer.2010.08.015