Backbone amide dynamics studies of apo-L75F-TrpR, a temperature sensitive mutant of the tryptophan repressor protein (TrpR): comparison with the 15N NMR … (original) (raw)

Abstract

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 corepressorbound) form. The 15 N 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 15 N NMR relaxation data ( 15 N-T 1 , 15 N-T 2 , and heteronuclear 15 N-{ 1 H}-nOe) recorded on all three aporepressors at a magnetic field strength of 600 MHz ( 1 H Larmor frequency) were analyzed to extract dynamics parameters, including diffusion tensor ratios (D ) /D^), correlation times (τ m ) for overall reorientations of the proteins in solution, reduced spectral density terms [J eff (0), J(0.87ω H ), J(ω 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 ) /D^ratios and characteristic time constants, τ m , for overall global reorientation, indicating that in solution, all three apoproteins display very similar overall shape, structure, and rotational diffusion properties. Comparison of 15 † multipulse sequence; CSA, chemical shift anisotropy; DIPSI, decoupling in the presence of scalar interactions; HSQC, heteronuclear single-quantum coherence spectroscopy; HTH, helix-turn-helix motif; IPTG, isopropyl β-thiogalactoside; 5-MT, 5-methyltryptophan; L-Trp, L-tryptophan; NMR, nuclear magnetic resonance; nOe, nuclear Overhauser effect; TrpR, tryptophan repressor; apo-WT-TrpR, wild-type apo-TrpR repressor; apo-L75F-TrpR, L75F mutant apo-TrpR repressor; A77V-TrpR, A77V mutant apo-TrpR repressor; ts, temperature-sensitive; WALTZ, wideband alternative-phase low-power technique for zero residual splitting.

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