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Abstract

Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of inter-nuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-τ_c (τ_c < supra-τ_c < 50 μs). Here we present a robust approach called Self-Consistent RDC-based Model-free analysis (SCRM) that delivers RDC-based order parameters—independent of the details of the structure used for alignment tensor calculation—as well as the dynamic average orientation of the inter-nuclear vectors in the protein structure in a self-consistent manner. For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors ⟨S²_rdc⟩=0.72±0.02 compared to ⟨S²_LS⟩ = 0.778 ± 0.003 for the Lipari–Szabo order parameters, indicating that the inclusion of the supra-τ_c window increases the averaged amplitude of mobility observed in the sub-τ_c window by about 34%. For the β-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S²_rdc(NH) = (0.59, 0.72, 0.59) was extracted. The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-τ_c time scale (S²_rdc(NH) = 0.59 ± 0.03), while it is inconspicuous (S²_LS(NH) = 0.82) on the sub-τ_c as well as on μs–ms relaxation dispersion time scales. The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.