Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 Å in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.

While extracting dynamics parameters from backbone 15N relaxation measurements in proteins has become routine over the past two decades, it is increasingly recognized that accurate quantitative analysis can remain limited by the potential presence of systematic errors associated with the measurement of 15N R1 and R2 or R1ρ relaxation rates as well as heteronuclear 15N-{1H} NOE values. We show that systematic errors in such measurements can be far larger than the statistical error derived from either the observed signal-to-noise ratio, or from the reproducibility of the measurement. Unless special precautions are taken, the problem of systematic errors is shown to be particularly acute in perdeuterated systems, and even more so when TROSY instead of HSQC elements are used to read out the 15N magnetization through the NMR-sensitive 1H nucleus. A discussion of the most common sources of systematic errors is presented, as well as TROSY-based pulse schemes that appear free of systematic errors to the level of < 1%. Application to the small perdeuterated protein GB3, which yields exceptionally high S/N and therefore is an ideal test molecule for detection of systematic errors, yields relaxation rates that show considerably less residue by residue variation than previous measurements. Measured R2′/R1′ ratios fit an axially symmetric diffusion tensor with a Pearson’s correlation coefficient of 0.97, comparable to fits obtained for backbone amide RDCs to the Saupe matrix.

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.

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822-5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098-6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible.

Detergents are often used to investigate the structure and dynamics of membrane proteins.W hereas the structural integrity seems to be preserved in detergents for many membrane proteins,t heir functional activity is frequently compromised, but can be restored in al ipid environment. Herein we showw ith per-residue resolution that while OmpX forms as table b-barrel in DPC detergent micelles,D HPC/ DMPC bicelles,a nd DMPC nanodiscs,t he pico-to nanosecond and micro-to millisecond motions differ substantially between the detergent and lipid environment. In particular for the b-strands,t here is pronounced dynamic variability in the lipid environment, whichappears to be suppressed in micelles. This unexpected complex and membrane-mimetic-dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein-protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme-substrate and protein-protein recognition. [1][2][3][4][5] Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein-protein recognition. [6,7] However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform.Nevertheless, for proteins and TAR-RNA, [8][9][10] recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques [11,12] have detected substantial internal dynamics in a time window from the rotational correlation time t c (one-digit nanoseconds) to approximately 50 ms, [8,[13][14][15] called the supra-t c window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined.Supra-t c dynamics in ubiquitin [9] and TAR-RNA [16] could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-t c motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-t c dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions.[1, 2, 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-t c window at room temperature.Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible.Significant motional amplitude in the supra-t c window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground