Backbone Dynamics of the Ribonuclease Binase Active Site Area Using Multinuclear (15N and 13CO) NMR Relaxation and Computational Molecular Dynamics

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Abstract
The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with 15N, 13C NMR relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics simulation. The data are compared with the temperature factors reported for the X-ray structure of this enzyme. The molecular dynamics and X-ray data correspond well and predict motions in the loops 56−61 and 99−104 that contain residues that specifically recognize substrate and are catalytic (His101), respectively. In contrast, the 15N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56−61 are evident from 13CO−13Cα cross relaxation data, but the mobility of the catalytic loop 99−104 is not detected by 13CO cross relaxation either. From the results of this and previous work [Wang, L., Pang, Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 7684−7689], the following dynamical characterization of the active site area of binase emerges: a beta sheet, rigid at all probed time scales, supports the catalytic residue Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow time scales, but the fast motions of the loop which contains the other catalytic residue, His 101, as predicted by B-factors and computational molecular dynamics is not detected by NMR relaxation. This work strongly argues for the use of several measures in the study of protein dynamics.