COMPARISON OF BACKBONE DYNAMICS OF REDUCED AND OXIDIZED ESCHERICHIA-COLI GLUTAREDOXIN-1 USING N-15 NMR RELAXATION MEASUREMENTS

NMR-based structure determination of Escherichia coli glutaredoxin-1 i n its reduced and oxidized forms revealed only subtle structural diffe rences between the two forms. In an effort to characterize the role dy namics may play in the functioning of the protein, the backbone dynami cs of both the reduced and oxidized forms of E. coli glutaredoxin-1 ha ve been characterized using inverse-detection two-dimensional N-15-H-1 NMR spectroscopy. Longitudinal (T-1) and transverse (T-2) N-15 relaxa tion time constants and steady-state {H-1}-N-15 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 (S-2), the effective correlation time for internal mo tions (tau(e)), N-15 exchange broadening contributions (R-ex), and the overall molecular rotational correlation time (tau(m)). Sedimentation equilibrium measurements showed the reduced protein to be monomeric w hereas 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 carrie d out at two concentrations with very different monomer/dimer ratios. There is increased motion on both nano-picosecond and micro-millisecon d time scales in the reduced form relative to the oxidized form, consi stent with a more rigid oxidized protein. The increase in motion in th e reduced protein correlates with its decreased thermodynamic stabilit y. 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.