De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.

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 15N backbone dynamics in differentially enriched 1-73(U-13C, 15N)/74-108(U-15N) reassembled thioredoxin on multiple timescales using a series of 2D and 3D MAS NMR experiments probing the backbone amide 15N longitudinal relaxation, 1H-15N dipolar order parameters, 15N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and 1H T2′ -filtered NCA experiments. The spin-lattice relaxation rates R1(R1 = 1/T1) 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 1H-15N dipolar order parameters, , and 15N CSA anisotropies, δσ reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average = 0.89 ± 0.06 and δσ = 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 α-helices and β-sheets, with no apparent systematic differences in dynamics between the α-helical and β-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.