Detection of the sulfhydryl groups in proteins with slow hydrogen exchange rates and determination of their proton/deuteron fractionation factors using the deuterium-induced effects on the 13C(beta) NMR signals.
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(beta) 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-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees 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 (13)C(beta) 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.
Takeda
Mitsuhiro
M
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
Jee
JunGoo
J
Terauchi
Tsutomu
T
Kainosho
Masatsune
M
D002247:Carbon Isotopes / Q000032:analysis; D003545:Cysteine / Q000032:analysis*; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011522:Protons
D002247:Carbon Isotopes; D011506:Proteins; D011522:Protons; D006859:Hydrogen; D003903:Deuterium; D003545:Cysteine