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Biochemistry. 1997 Apr 22;36(16):5029-44.

Comparison of backbone dynamics of reduced and oxidized Escherichia coli glutaredoxin-1 using 15N NMR relaxation measurements.

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  • 1Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, USA.

Abstract

NMR-based structure determination of Escherichia coli glutaredoxin-1 in its reduced and oxidized forms revealed only subtle structural differences between the two forms. In an effort to characterize the role dynamics may play in the functioning of the protein, the backbone dynamics of both the reduced and oxidized forms of E. coli glutaredoxin-1 have been characterized using inverse-detection two-dimensional 15N-1H NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state [1H]-15N NOEs were measured for a majority of the protonated backbone nitrogen atoms. These data were analyzed by using a model-free formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau(e)), 15N exchange broadening contributions (R(ex)), and the overall molecular rotational correlation time (tau(m)). Sedimentation equilibrium measurements showed the reduced protein to be monomeric whereas the oxidized form could be fit to a monomer-dimer equilibrium. In order to try and assess the effect of dimerization on the dynamical parameters, the measurements on the oxidized protein have been carried out at two concentrations with very different monomer/dimer ratios. There is increased motion on both nano-picosecond and micro-millisecond time scales in the reduced form relative to the oxidized form, consistent with a more rigid oxidized protein. The increase in motion in the reduced protein correlates with its decreased thermodynamic stability. The role of the observed differences in the dynamic behavior in the two forms, particularly in the active site, in glutaredoxin-1's role as a protein disulfide reductant is discussed.

PMID:
9125525
DOI:
10.1021/bi962181g
[PubMed - indexed for MEDLINE]
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