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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dc.contributor.authorTreweek, TMen_AU
dc.contributor.authorRekas, Aen_AU
dc.contributor.authorWalker, MJen_AU
dc.contributor.authorCarver, JAen_AU
dc.date.accessioned2011-09-20T23:16:49Zen_AU
dc.date.available2011-09-20T23:16:49Zen_AU
dc.date.issued2010-11-01en_AU
dc.date.statistics2011-09-20en_AU
dc.description.abstractThe 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.en_AU
dc.identifier.citationTreweek, 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.2010.08.015en_AU
dc.identifier.govdoc3470en_AU
dc.identifier.issn0014-4835en_AU
dc.identifier.issue5en_AU
dc.identifier.journaltitleExperimental Eye Researchen_AU
dc.identifier.pagination691-699en_AU
dc.identifier.urihttp://dx.doi.org/10.1016/j.exer.2010.08.015en_AU
dc.identifier.urihttp://apo.ansto.gov.au/dspace/handle/10238/3755en_AU
dc.identifier.volume91en_AU
dc.language.isoenen_AU
dc.publisherElsevieren_AU
dc.subjectHeat-shock proteinsen_AU
dc.subjectQuantitative chemical analysisen_AU
dc.subjectNMR spectraen_AU
dc.subjectPolypeptidesen_AU
dc.subjectCrystalline lensen_AU
dc.titleA quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallinen_AU
dc.typeJournal Articleen_AU
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