Thermal Stability of Calmodulin and Mutants Studied by 1H-15N HSQC NMR Measurements of Selectively Labeled [15N]Ile Proteins
详细信息 查看全文
- 作者：Rodolfo R. Biekofsky ; Stephen R. Martin ; John E. McCormick ; Laura Masino ; Sandrine Fefeu ; Peter M. Bayley ; and James Feeney
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 28, 2002
- 年：2002
- 卷：41
- 期：21
- 页码：6850 - 6859
- 全文大小：144K
- 年卷期：v.41,no.21(May 28, 2002)
- ISSN：1520-4995
文摘
Calmodulin, the Ca2+-dependent activator of many cellular processes, contains two well-definedstructural domains, each of which binds two Ca2+ ions. In its Ca2+-free (apo) form, it provides an attractivemodel for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes,indicating two structurally autonomous folding units. 1H-15N HSQC NMR in principle offers a detailedpicture of the behavior of individual residues during protein unfolding transitions, but is limited by thelack of dispersion of resonances in the unfolded state. In this work, we have used selective [15N]Ile labelingof four distinctive positions in each calmodulin domain to monitor the relative thermal stability of thefolding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized.These mutations lead to a characteristic perturbation of the stability (Tm) of the nonmutated domain relativeto that of wild-type apocalmodulin. The ability to monitor specific 15N-labeled residues, well-distributedthroughout the domain, provides strong evidence for the autonomy of a given folding unit, as well asproviding accurate measurements of the unfolding parameters Tm and 
lta.gif" BORDER=0 >Hm. The thermodynamic parametersare interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin,where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changesin the observed unfolding behavior of a given folding unit. The selective 15N labeling approach is thus ageneral method that can provide detailed information about structural intermediates populated in complexprotein unfolding processes.
lta.gif" BORDER=0 >Hm. The thermodynamic parametersare interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin,where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changesin the observed unfolding behavior of a given folding unit. The selective 15N labeling approach is thus ageneral method that can provide detailed information about structural intermediates populated in complexprotein unfolding processes.