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

Abstract

Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of internuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-s c (s c \ supra-s c \ 50 ls). Here we present a robust approach called Self-Consistent RDC-based Model-free analysis (SCRM) that delivers RDC-based order parametersindependent 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 S 2 rdc ¼ 0:72 AE 0:02 compared to S 2 LS = 0.778 ± 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-s c window increases the averaged amplitude of mobility observed in the sub-s c window by about 34%. For the b-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S 2 rdc ðNHÞ = (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-s c time scale (S 2 rdc ðNHÞ = 0.59 ± 0.03), while it is inconspicuous (S 2 LS ðNHÞ = 0.82) on the sub-s c as well as on ls-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.

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

“…As shown in many works, 0.5 to 1 μs-long MD simulations of ubiquitin can reproduce NMR order parameters and residual dipolar couplings very well, indicating good agreement between simulations and experimental observables. 16,69,72,90 Also, such simulations reproduce local and large-scale conformational fluctuations observed in X-rays structures. The latter are easily visualized by projecting the MD trajectory on a two-dimensional plane built from available X-rays structures as shown in a number of recent works.…”

Intermolecular dynamics studied by paramagnetic tagging

“…The RDC is also sensitive to motions spanning the ps-ms timescale. Thus, the dynamics information obtained from RDCs offer a view on dynamic processes that is complementary to other NMR spectroscopic approaches (Tolman and Ruan 2006;Lakomek et al 2008;Lange et al 2008).…”

How to tackle protein structural data from solution and solid state: An integrated approach

“…In this example, H N -N RDCs measured from as many as 36 different alignment media [54,103] were used [94] for the refinement of an X-ray structure of ubiquitin (PDB code: 3NHE), which displayed a 1.26 Å resolution. The initial disagreement shown for the original structure decreases, in terms of Q RDC , from 0.360 to 0.121 after performing REFMAC-NMR refinement (Fig.…”

“…In turn, partial alignment prevents internuclear dipolar interactions to be completely abolished by rotation, causing RDCs. This self-orientation is an alternate way to generate RDCs without using an external alignment medium [4,[42][43][44][45][46][47][48][49][50][51][52][53][54]. PCSs and paramagnetic RDCs depend on the molecular nuclear coordinates in a common frame defined by the magnetic susceptibility anisotropy tensor associated with the paramagnetic metal.…”