Self-consistent residual dipolar coupling based model-free analysis for the robust determination of nanosecond to microsecond protein dynamics

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-[Formula: see text] ([Formula: see text] < supra-[Formula: see text] < 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 [Formula: see text] compared to [Formula: see text] = 0.778 ± 0.003 for the Lipari–Szabo order parameters, indicating that the inclusion of the supra-[Formula: see text] window increases the averaged amplitude of mobility observed in the sub-[Formula: see text] window by about 34%. For the β-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter [Formula: see text] = (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-[Formula: see text] time scale ([Formula: see text] = 0.59 ± 0.03), while it is inconspicuous ([Formula: see text] = 0.82) on the sub-[Formula: see text] 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-008-9244-4) contains supplementary material, which is available to authorized users.