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.

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.

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.

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

This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098-6107, 2001; Lakomek in J Biomol NMR 34: [101][102][103][104][105][106][107][108][109][110][111][112][113][114][115] 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supras c dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time s c . In fact, the average amplitude of motion expressed in terms of order parameters S 2 associated with the supra-s c window brings evidence to the existence of fluctuations contributing as much additional mobility as those already present in the faster ps-ns time scale measured from relaxation data. Comparison to previous results on ubiquitin demonstrates that the RDC-derived order parameters are dominated both by rotameric interconversions and faster libration-type motions around equilibrium positions. They match best with those derived from a combined J-coupling and residual dipolar coupling approach (Chou in J Am Chem Soc 125:8959-8966, 2003) taking backbone motion into account. In order to appreciate the dynamic scale of side chains over the entire protein, the methyl group order parameters are compared to existing dynamic ensembles of ubiquitin. Of those recently published, the broadest one, namely the EROS ensemble (Lange in Science 320: [1471][1472][1473][1474][1475] 2008), fits the collection of methyl group order parameters presented here best. Last, we used the MFAderived averaged spherical harmonics to perform highlyparameterized rotameric searches of the side chains conformation and find expanded rotamer distributions with excellent fit to our data. These rotamer distributions suggest the presence of concerted motions along the side chains.

A robust procedure for the determination of protein-backbone motions on time scales of pico- to milliseconds directly from residual dipolar couplings has been developed that requires no additional scaling relative to external references. The results for ubiquitin (blue in graph: experimental N-HN order parameters) correspond closely to the amplitude, nature, and distribution of motion found in a 400 ns molecular-dynamics trajectory of ubiquitin (red).

Ein zuverlässiges Verfahren zur Bestimmung der Bewegungen des Proteingerüsts über einen Zeitraum von Piko‐ bis Millisekunden auf der Grundlage dipolarer Restkopplungen erfordert keine Skalierung mithilfe einer externen Referenz. Die Ergebnisse für Ubiquitin (blau im Graph: experimentelle N‐HN‐Ordnungsparameter) entsprechen in Amplitude, Art und Verteilung sehr gut der Bewegung, die in einer 400‐ns‐Moleküldynamiktrajektorie von Ubiquitin ermittelt wurde (rot).