Abstract We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with 15N R 1, R 1Ï? , and {1H}-15N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo â??gatingâ?? motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant Rex contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the nsâ??ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.
Content Type Journal Article
Category Article
Pages 71-82
DOI 10.1007/s10858-011-9542-0
Authors
Teresa Miletti, Department of Chemistry, McGill University, Montreal, QC H3A 2K6, Canada
Patrick J. Farber, Department of Chemistry, McGill University, Montreal, QC H3A 2K6, Canada
Anthony Mittermaier, Department of Chemistry, McGill University, Montreal, QC H3A 2K6, Canada
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J Mol Biol. 2005 May 27;349(1):163-83
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Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by...
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11-25-2010 08:21 PM
[NMR paper] NMR structure of the ribosomal protein L23 from Thermus thermophilus.
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The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23...
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11-24-2010 09:01 PM
[NMR paper] Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15
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11-18-2010 08:31 PM
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08-22-2010 05:08 PM
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2H NMR Spin Relaxation for studying RNA dynamics
A Suite of 2H NMR Spin Relaxation Experiments for the Measurement of RNA Dynamics
Pramodh Vallurupalli and Lewis E. Kay, J. Am. Chem. Soc.; 2005; 127 (18) pp 6893 - 6901
ABSTRACT:
A suite of (2)H-based spin relaxation NMR experiments is presented for the measurement of molecular dynamics in a site-specific manner in uniformly (13)C, randomly fractionally deuterated ( approximately 50%) RNA molecules. The experiments quantify (2)H R(1) and R(2) relaxation rates that can subsequently be analyzed to obtain information about dynamics on a pico- to nanosecond time scale. Sensitivity...
Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation
Linear Mode
