PMC:2728246 / 2142-7617 JSONTXT

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Initial studies based on backbone amide relaxation and relaxation dispersion methods have elucidated motional processes in the protein backbone occurring on the fast time scale (\u003cτc, ns) as well as the slower time window associated with chemical exchange (Kay et al. 1989; Mandel et al. 1996; Palmer et al. 2001).\nSimilarly to the residue-specific investigation of mobility in the backbone, side chain disorder has also been studied with residue resolution as a result of development of NMR methodologies targeting methyl groups. Measurement of relaxation rates of 13C and 2H in isotope-labeled methyl groups are now being used routinely to understand side chain dynamics (Lee et al. 1999; Schneider et al. 1992; Igumenova et al. 2006; Tugarinov and Kay 2005, 2006; Xue et al. 2007), while methods for the quantitative interpretation of side chain order parameters are still being improved (Meirovitch et al. 2006; Showalter et al. 2007). Solid-state NMR relaxation techniques have also been used to study methyl group dynamics and suggested a high concordance between the motions for a protein in solution and in the aggregated state (Agarwal et al. 2008; Reif et al. 2006). Recent interests in the hydrophobic mechanism of folding, packing and recognition of protein-ligand, protein-protein and other protein assemblies have used 2H relaxation rates in methyl groups to describe their flexibility (Mulder et al. 2002; Skrynnikov et al. 2001; Mittermaier and Kay 2002; Korzhnev et al. 2004; Eisenmesser et al. 2005; Tugarinov and Kay 2006) and residual conformation entropy of side chains (Lee et al. 2000; Frederick et al. 2006; Li and Brüschweiler 2009).\nAn inherent drawback of relaxation-based dynamics, however, is their limitation to time scales faster than the correlation time (\u003cτc, ns) and within the chemical shift exchange (μs–ms) time window, leaving the so-called supra-τc time scale between ns and μs, believed to partially enclose folding-unfolding processes as well as conformational sampling, largely invisible to such measurements.\nNevertheless, successful observations of supra-τc motions in side chains were possible by directly measuring the dynamic averaging of NMR interaction observables. The reduction of 13C–1H dipolar couplings in side chains were measured using solid-state NMR approaches in aggregated proteins (ubiquitin and Pf1 coat protein) and revealed motions faster than the μs time scale (Lorieau and McDermott 2006; Lorieau et al. 2008). Also, side chain rotameric interconversions up to ms were suggested based on residual dipolar couplings (RDC) measured in the side chains (Mittermaier and Kay 2001). In related fashion, using a combined approach of J-coupling and RDC measurements, a description of χ1 rotamer dynamics for protein G and ubiquitin was possible (Chou et al. 2003).\nIndeed, RDCs have been recognized to provide a unique perspective on the internal structural fluctuations of proteins even over time scales slower than the correlation time and have thus emerged as the method of choice for studying protein backbone motions in the supra-τc time window (Meiler et al. 2001; Tolman 2001, 2009; Lakomek et al. 2005, 2008a; Bouvignies et al. 2005; Yao et al. 2008; Salmon et al. 2009; Ho and Agard 2009). The relevance of the dynamics occurring in this range was recently highlighted using RDCs, demonstrating that the conformational space attained by human ubiquitin was sufficient to explain the selection of all its known binding partners (Lange et al. 2008). The model-free description of RDC-based dynamics now benefits from robust analysis methods which assess the self-consistency of the dataset and circumvent the pitfall of structural noise (Lakomek et al. 2008a).\nInterestingly, backbone amide RDC analysis has revealed a connection between backbone and side chain dynamics by demonstrating a correlation of the dynamics with the solvent accessibility of the side chain (Lakomek et al. 2005). The prospect of side chains transmitting their motion to the backbone can be rationalised by considering that the sampling of the rotameric energetic wells for all χn torsion angles along the side chain will cause substantial structural changes which may require some backbone accommodation to avoid steric clashes.\nBy extending the established approaches to side chains, this work aims to gain deeper insights into the complex fluctuations of side chains in the supra-τc time window and their relationship with backbone dynamics. To this end, we present a model-free analysis (MFA) on a large RDC dataset measured from the side-chain methyl groups of ubiquitin (Lange et al. 2008, Table S7). Our results show a large amount of conformational dynamics of the CCmet bond extending into the supra-τc time window. We present a careful examination of these fluctuations, assessing the influence of side chain length, residue type and solvent exposure, and evaluating their relationship to previous results. This approach promises to contribute to recent interests in side chain dynamics, particularly to understand the mechanism of folding, packing and recognition of proteins."}