A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallin

作者：Treweek Teresa M; Rekas Agata; Walker Mark J; Carver John A^*

来源：Experimental Eye Research, 2010, 91(5): 691-699.

DOI：10.1016/j.exer.2010.08.015

• small heat-shock protein
• molecular chaperone
• NMR Spectroscopy
• C-terminal extension
• site directed mutagenesis

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 (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, alpha A- and alpha B-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 alpha A- and alpha B-crystallin in the absence and presence of a bound target protein, reduced alpha-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 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 (1)H-(15)N 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.

• 出版日期2010-11