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Type: | Journal article |
Title: | A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, αA- and αB-crystallin |
Other Titles: | A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallin |
Author: | Treweek, T. Rekas, A. Walker, M. Carver, J. |
Citation: | Experimental Eye Research, 2010; 91(5):691-699 |
Publisher: | Academic Press Ltd |
Issue Date: | 2010 |
ISSN: | 0014-4835 1096-0007 |
Statement of Responsibility: | Teresa M. Treweek, Agata Rekas, Mark J. Walker, John A. Carver |
Abstract: | The principal lens proteins αA- and αB-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, αA- and αB-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. αB-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either αA- or αB-crystallin is not known nor is the mechanism of their chaperone action. Our earlier (1)H 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, αA- and αB-crystallin were (15)N-labelled and their (1)H-(15)N through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. (1)H-(15)N spin-lattice (T(1)) and spin-spin (T(2)) relaxation times were measured for αA- and αB-crystallin in the absence and presence of a bound target protein, reduced α-lactalbumin. (1)H-(15)N 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 αA- and αB-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of αA-crystallin with reduced α-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of αB-crystallin in its C-terminal region: (i) I159A and I161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One 2, e1046). Comparison of (1)H-(15)N NOE values for these mutants with wild type αB-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L αB-crystallin, which may affect chaperone ability. |
Keywords: | small heat-shock protein molecular chaperone NMR Spectroscopy C-terminal extension site directed mutagenesis |
Rights: | Copyright © 2010 Elsevier Ltd. All rights reserved. |
DOI: | 10.1016/j.exer.2010.08.015 |
Grant ID: | ARC |
Published version: | http://dx.doi.org/10.1016/j.exer.2010.08.015 |
Appears in Collections: | Aurora harvest 5 Chemistry and Physics publications IPAS publications |
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