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[摘要]：Backbone amide dynamics studies were conducted on a temperature-sensitive mutant (L75F-TrpR) of the tryptophan repressor protein (TrpR) of Escherichia coli in its apo (i.e., no L-tryptophan corepressor-bound) form. The N-15 NMR relaxation profiles of apo-L75F-TrpR were analyzed and compared to those of wild-type (WT) and super-repressor mutant (A77V) TrpR proteins, also in their apo forms. The N-15 NMR relaxation data (N-15-T-1, N-15-T-2, and heteronuclear N-15-{H-1}-nOe) recorded on all three aporepressors at a magnetic field strength of 600 MHz (H-1 Larmor frequency) were analyzed to extract dynamics parameters, including diffusion tensor ratios (D-parallel to/D-perpendicular to), correlation times (tau(m)) for overall reorientations of the proteins in solution, reduced spectral density terms [J(eff)(0), J(0.87 omega(H)), J(omega(N))], and generalized order parameters (S-2), which report on protein internal motions on the picosecond to nanosecond and slower microsecond to millisecond chemical exchange time scales. Our results indicate that all three aporepressors exhibit comparable D-parallel to/D-perpendicular to ratios and characteristic time constants, tau(m), for overall global reorientation, indicating that in solution, all three apoproteins display very similar overall shape, structure, and rotational diffusion properties. Comparison of N-15 NMR relaxation data, reduced spectral density profiles, and generalized S-2 order parameters indicated that these parameters are quite uniform for backbone amides positioned within the four (A-C and F) core a-helices of all three aporepressors. In contrast, small but noticeable differences in internal dynamics were observed for backbone amides located within the helix D-turn-helix E DNA-binding domain of the apo-TrpR proteins. The significance of these dynamics differences in terms of the biophysical characteristics and ligand binding properties of the three apo-TrpR proteins is discussed. |
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