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

Journal Article


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 1H 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, αA- and αB-crystallin were 15N-labelled and their 1H-15N through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. 1H-15N spin-lattice (T1) and spin-spin (T2) relaxation times were measured for αA- and αB-crystallin in the absence and presence of a bound target protein, reduced α-lactalbumin. 1H-15N 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 extension: (i) I159A and I161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One, 2, e1046). Comparison of 1H-15N 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.

Authors


  •   Treweek, Teresa M.
  •   Rekas, Agata (external author)
  •   Walker, Mark J. (external author)
  •   Carver, John A. (external author)

Publication Date


  • 2010

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, alphaA- and alphaB-crystallin. Experimental Eye Research, 91 (5), 691-699.

Scopus Eid


  • 2-s2.0-78049506985

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3971

Number Of Pages


  • 8

Start Page


  • 691

End Page


  • 699

Volume


  • 91

Issue


  • 5

Place Of Publication


  • www.elsevier.com/locate/yexer

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 1H 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, αA- and αB-crystallin were 15N-labelled and their 1H-15N through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. 1H-15N spin-lattice (T1) and spin-spin (T2) relaxation times were measured for αA- and αB-crystallin in the absence and presence of a bound target protein, reduced α-lactalbumin. 1H-15N 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 extension: (i) I159A and I161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One, 2, e1046). Comparison of 1H-15N 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.

Authors


  •   Treweek, Teresa M.
  •   Rekas, Agata (external author)
  •   Walker, Mark J. (external author)
  •   Carver, John A. (external author)

Publication Date


  • 2010

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, alphaA- and alphaB-crystallin. Experimental Eye Research, 91 (5), 691-699.

Scopus Eid


  • 2-s2.0-78049506985

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/3971

Number Of Pages


  • 8

Start Page


  • 691

End Page


  • 699

Volume


  • 91

Issue


  • 5

Place Of Publication


  • www.elsevier.com/locate/yexer