TY - JOUR
T1 - Probing the Flexibility of the DsbA Oxidoreductase from Vibrio cholerae-a 15N - 1H Heteronuclear NMR Relaxation Analysis of Oxidized and Reduced Forms of DsbA
AU - Horne, Henry James
AU - d'Auvergne, Edward J
AU - Coles, Murray
AU - Velkov, Tony
AU - Chin, Yanni
AU - Charman, William Neil
AU - Prankerd, Richard John
AU - Gooley, Paul
AU - Scanlon, Martin
PY - 2007
Y1 - 2007
N2 - We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. the reduced structure shoes a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helocal domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerbaly more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike s information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the oico- to nanosecond and micro- to millisecond timescale. many significant changes in dynamics are located either close to the active site or at the intersection points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect affect the catalytic cleft of the enzyme. taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.
AB - We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. the reduced structure shoes a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helocal domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerbaly more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike s information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the oico- to nanosecond and micro- to millisecond timescale. many significant changes in dynamics are located either close to the active site or at the intersection points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect affect the catalytic cleft of the enzyme. taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.
M3 - Article
SN - 0022-2836
VL - 371
SP - 703
EP - 716
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -