Gd(III) Chelates as NMR Probes of Protein鈥揚rotein Interactions. Case Study: Rubredoxin and Cytochrome c3
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- 作者：Rui M. Almeida ; Carlos F. G. C. Geraldes ; Sofia R. Pauleta ; Jos茅 J. G. Moura
- 刊名：Inorganic Chemistry
- 出版年：2011
- 出版时间：November 7, 2011
- 年：2011
- 卷：50
- 期：21
- 页码：10600-10607
- 全文大小：988K
- 年卷期：v.50,no.21(November 7, 2011)
- ISSN：1520-510X
文摘
Two cyclen-derived Gd probes, [Gd鈥揇OTAM]3+ and [Gd鈥揇OTP]5鈥?/sup> (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein鈥損rotein interactions. Two proteins, Desulfovibrio gigas rubredoxin and Desulfovibrio gigas cytochrome c3, were used as model partners. In a 1H NMR titration it was shown that [Gd鈥揇OTP]5鈥?/sup> binds to cytochrome c3 near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A Kd of 23 卤 1 渭M was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd鈥揇OTP]5鈥?/sup> binding to cytochrome c3 at a molar ratio of 2. The other probe, [Gd鈥揇OTAM]3+, caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19鈥揋23 and W37鈥揝45, which broaden beyond detection). This effect was partially reversed for some resonances (C6鈥揧11, in particular) when cytochrome c3 was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners鈥?binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface.