Abstract
<jats:p>Dynamics in proteins occur on a wide range of timescales and are crucial for protein function. On the fast end of that timescale, pico- to nanosecond dynamics have been extensively employed as proxies for entropy and their amplitude can be described by order parameters. Experimentally, NMR can be used to determine order parameters of the protein backbone and, via deuterium relaxation, of methyl groups, yet such experiments cannot be applied to large protein assemblies. In contrast, relaxation-violated coherence transfer experiments, that allow for the determination of side chain order parameters in highly deuterated, methyl-labeled proteins, are more sensitive. Here, we demonstrate that such experiments can be applied to very large, asymmetric protein assemblies by determining axial methyl order parameters for the 300 kDa fully asymmetric core of the eukaryotic RNA exosome complex. Ile-δ1[13CH3] methyl groups adopt a wide range of order parameters but highly flexible side chains are infrequent. High quality data, which we obtain for flexible regions, is required to observe subtle effects of RNA binding on order parameters. Local cryo-EM Q-scores correlate moderately with order parameters suggesting that Q-scores contain information on nanosecond motions. AF2χ, a recently described prediction tool for side-chain variability, provides good estimates of methyl order parameters, which are, in favorable cases, strongly correlated with experimental values. We thus demonstrate that relaxation-violated coherence transfer experiments can be employed to determine order parameters in large, asymmetric protein complexes that are difficult to capture by other methods, yet are crucial for the understanding of protein function.</jats:p>