Preparation of spin-labeled bacterial ribonuclease from Bacillus intermedius 7P for EPR studies of protein dynamics.
The optimal conditions for labeling of binase (a bacterial ribonuclease isolated from Bacillus intermedius 7P) with a bromoacetamide spin label have been determined. The label is suitable for probing the dynamics of the protein by electron paramagnetic resonance (EPR). Binase samples specifically labeled at residue His-101 of the active center were prepared by incubation for 48 h at 20 degrees C in potassium phosphate buffer (pH 5.5) containing the bromoacetamide (1:3 protein/label molar ratio). Fluorescence assay showed that the structure of the labeled binase is indistinguishable from that of the native protein.
Yagodina
L O
LO
Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, Kazan, 420503, Russia. yagodina@sci.kcn.ru
Altshuler
A E
AE
Abreimova
Y V
YV
D001407:Bacillus / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D013113:Spin Labels; D004722:Endoribonucleases; C022543:ribonuclease T(2)