Preparation of spin-labeled bacterial ribonuclease from Bacillus intermedius 7P for EPR studies of protein dynamics. |
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Authors: | L O Yagodina A E Altshuler Y V Abreimova |
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Institution: | Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, Kazan, 420503, Russia. yagodina@sci.kcn.ru |
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Abstract: | 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. |
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