A quantitative NMR spectroscopic examination of the flexibility of the C-terminal ext
 

A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alphaA- and alphaB-crystallin.

Related Articles A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alphaA- and alphaB-crystallin.

Exp Eye Res. 2010 Aug 20;

Authors: Treweek TM, Rekas A, Walker MJ, Carver JA

The principal lens proteins alphaA- and alphaB-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, alphaA- and alphaB-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. alphaB-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either alphaA- or alphaB-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, J.A.(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, alphaA- and alphaB-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 alphaA- and alphaB-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 alphaA- and alphaB-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of alphaA-crystallin with reduced alpha-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of alphaB-crystallin in its C-terminal extension: (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 alphaB-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L alphaB-crystallin, which may affect chaperone ability.

PMID: 20732317 [PubMed - as supplied by publisher]



Source: PubMed