Preparation of Spin-Labeled Bacterial Ribonuclease from Bacillus intermedius 7P for EPR Studies of Protein Dynamics

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°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.