Backbone amide dynamics studies of apo-L75F-TrpR, a temperature sensitive mutant of the tryptophan repressor protein (TrpR): comparison with the 15N NMR relaxation profiles of wild type and A77V mutant apo-TrpR repressors.
Related Articles Backbone amide dynamics studies of apo-L75F-TrpR, a temperature sensitive mutant of the tryptophan repressor protein (TrpR): comparison with the 15N NMR relaxation profiles of wild type and A77V mutant apo-TrpR repressors.
Biochemistry. 2010 Aug 18;
Authors: Goel A, Tripet BP, Tyler RC, Nebert LD, Copie V
Backbone amide dynamics studies were conducted on a temperature sensitive mutant (L75F-TrpR) of the tryptophan repressor protein (TrpR) of E. coli in its apo (i.e. no L-tryptophan co-repressor-bound) form. The 15N 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 15N NMR relaxation data (15N-T1, 15N-T2 and heteronuclear 15N-{1H}-nOe) recorded on all three apo-repressors at a magnetic field strength of 600 MHz (1H Larmor frequency) were analyzed to extract dynamics parameters including diffusion tensor ratios (D||/D perpendicular), correlation times (taum) for overall reorientations of the proteins in solution, reduced spectral density terms (Jeff(0), J(0.87omegaH), J(omegaN)) and generalized order parameters (S2), which report on protein internal motions on the ps-ns and slower mus-ms chemical exchange timescales. Our results indicate that all three apo-repressors exhibit comparable D||/D perpendicular ratios and characteristic time constants, taum, for overall global reorientation, indicating that in solution, all three apo-proteins display very similar overall shape, structure, and rotational diffusion properties. Comparison of 15N NMR relaxation data, reduced spectral density profiles, and generalized S2 order parameters indicated that these parameters are quite uniform for backbone amides positioned within the four (A, B, C, and F) core alpha-helices of all three apo-repressors. 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.
PMID: 20718459 [PubMed - as supplied by publisher]
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PubMed