Maria Luisa Tasayco

Dynamics of reassembled thioredoxin studied by magic angle spinning NMR: snapshots from different time scales.

Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this study, we report (15)N backbone dynamics in differentially enriched 1-73(U-(13)C,(15)N)/74-108(U-(15)N) reassembled thioredoxin on multiple time scales using a series of 2D and 3D MAS NMR experiments probing the backbone amide (15)N longitudinal relaxation, (1)H-(15)N dipolar order parameters, (15)N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and (1)H T(2)-filtered NCA experiments. The spin-lattice relaxation rates R(1) (R(1) = 1/T(1)) were observed in the range from 0.012 to 0.64 s(-1), indicating large site-to-site variations in dynamics on pico- to nanosecond time scales. The (1)H-(15)N dipolar order parameters, , and (15)N CSA anisotropies, delta(sigma), reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average = 0.89 +/- 0.06 and delta(sigma) = 92.3 +/- 5.2 ppm, respectively. From the aggregate of experimental data from different dynamics methods, some degree of correlation between the motions on the different time scales has been suggested. Analysis of the dynamics parameters derived from these solid-state NMR experiments indicates higher mobilities for the residues constituting irregular secondary structure elements than for those located in the alpha-helices and beta-sheets, with no apparent systematic differences in dynamics between the alpha-helical and beta-sheet residues. Remarkably, the dipolar order parameters derived from the solid-state NMR measurements and the corresponding solution NMR generalized order parameters display similar qualitative trends as a function of the residue number. The comparison of the solid-state dynamics parameters to the crystallographic B-factors has identified the contribution of static disorder to the B-factors. The combination of longitudinal relaxation, dipolar order parameter, and CSA line shape analyses employed in this study provides snapshots of dynamics and a new insight on the correlation of these motions on multiple time scales.

Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies.

Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.