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• 作者：Mitsuhiro Takeda ; JunGoo Jee ; Tsutomu Terauchi ; Masatsune Kainosho
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：May 5, 2010
• 年：2010
• 卷：132
• 期：17
• 页码：6254-6260
• 全文大小：363K
• 年卷期：v.132,no.17(May 5, 2010)
• ISSN：1520-5126

文摘

A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C_β chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis−trans isomerase b (EPPIb), which was selectively labeled with [3-¹³C;3,3-²H₂]Cys, showed much narrower line widths for the ¹³C_β NMR signals, as compared to those of the proteins labeled with either [3-¹³C]Cys or (3R)-[3-¹³C;3-²H]Cys. The ¹³C_β signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-¹³C;3,3-²H₂]Cys, split into four signals in H₂O/D₂O (1:1) at 40 °C and pH 7.5, indicating that the exchange rates of the side-chain SH’s and the backbone amides are too slow to average the chemical shift differences of the ¹³C_β signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4−0.5 for [SD]/[SH] and 0.9−1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE’s of the two slowly exchanging SH’s were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.