Dynamical Mapping of E. coli Thioredoxin via 13C NMR Relaxation Analysis

Abstract

NMR relaxation analysis was used to characterize the internal dynamics of oxidized E. coli thioredoxin in both the picosecond-nanosecond and microsecond-millisecond frequency ranges for 413 H−C and H−N bond vectors. The 13 C relaxation data was obtained utilizing protein samples possessing an alternating 13 C− 12 C− 13 C... labeling pattern for most enriched sites. When combined with partial deuteration, this labeling pattern provides for isolated 1 H− 13 C IS spin pairs exhibiting dynamically interpretable relaxation behavior. Side chains were found to exhibit a far broader range of dynamics than have been previously characterized for main chain resonances. The dynamics of structurally buried aromatic and leucine side chains are interpreted in terms of correlated main chain-side chain torsional oscillations. Structural regions exhibiting millisecond dynamics were found to correlate strongly with the presence of side chain-main chain or bifurcated main chain hydrogen bonds. Nuclei around the active site disulfide that exhibit mobility in the millisecond range correspond closely to the set of nuclei whose chemical shifts are altered upon reduction. The transient conformation is interpreted in terms of enhanced reactivity due to strain at the disulfide linkage.