Inhibition of [(15)N]valine transamination during selective labeling of barstar in a T7 polymerase system.

Selective labeling of barstar by the stable (15)N isotope of the valine residue with high selectivity of the label incorporation resulting from the process of gene expression in Escherichia coli BL21(DE3) has been optimized. We have shown that alpha-aminooxyacetic acid (AOAA) significantly reduces the isotope redistribution, thus increasing the selectivity of (15)N incorporation into the synthesized protein, as detected by 2D-NMR. Quantitative measurements were used to determine the selectivity for the incorporation of isotope-labeled valine residue, which was 96% in the case using AOAA. Studies of the dynamics of barstar synthesis showed that no suppression of barstar yield is observed under the regulation of the T7 polymerase expression system by isopropylthio-beta-D-galactoside (IPTG) and rifampicin using AOAA.

Interaction of the bacterial ribonuclease binase with the polypeptide inhibitor barstar based on kinetic data on poly(U) hydrolysis.

The reaction of poly(U) hydrolysis catalyzed by binase while the latter is inhibited by barstar has been investigated. The inhibition constant for barstar and the apparent Michaelis constants for the inhibition by barstar in the presence of ethanol and NaCl have been determined. Both ethanol and NaCl enhance the inhibition by barstar. This suggests that the binding of barstar with binase is probably due to the interaction of hydrophobic sites rather than by electrostatic interaction between amino acid residues.

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.

Superslow backbone protein dynamics as studied by 1D solid-state MAS exchange NMR spectroscopy.

Superslow backbone dynamics of the protein barstar and the polypeptide polyglycine was studied by means of a solid-state MAS 1D exchange NMR method (time-reverse ODESSA) that can detect reorientation of nuclei carrying anisotropic chemical shift tensors. Experiments were performed on carbonyl 13C in polyglycine (natural abundance) and backbone 15N nuclei in uniformly 15N-enriched barstar within a wide range of temperatures in dry and wet powders for both samples. Two exchange processes were observed in the experiments: molecular reorientation and spin diffusion. Experimental conditions that are necessary to separate these two processes are discussed on a quantitative level. It was revealed that the wet protein undergoes molecular motion in the millisecond range of correlation times, whereas in dry protein and polyglycine molecular reorientations could not be detected. The correlation time of the motion in the wet barstar at room temperature is 50-100 ms; the activation energy is about 80 kJ/mol. Previously, protein motions with such a long correlation time could be observed only by methods detecting chemical exchange in solution (e.g., hydrogen exchange). The application of solid-state MAS exchange spectroscopy provides new opportunities in studying slow biomolecular dynamics that is important for the biological function of proteins.