BACKBONE DYNAMICS OF THE BACILLUS-SUBTILIS GLUCOSE PERMEASE-IIA DOMAIN DETERMINED FROM N-15 NMR RELAXATION MEASUREMENTS

The backbone dynamics of the uniformly N-15-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional H-1-N-15 NMR spectroscopy. Longitudinal (T1) and transverse (T2) N-15 relaxation time constants and steady-state {H-1}-N-15 NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau(e)), and N-15 exchange broadening contributions (R(ex)) for each residue, as well as the overall molecular rotational correlation time (tau(m)). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, {H-1}-N-15 NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau(e) values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.