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Detection of nanosecond time scale side-chain jumps in a protein dissolved in water/glycerol solvent

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Abstract

In solution, the correlation time of the overall protein tumbling, τ R , plays a role of a natural dynamics cutoff—internal motions with correlation times on the order of τ R or longer cannot be reliably identified on the basis of spin relaxation data. It has been proposed some time ago that the ‘observation window’ of solution experiments can be expanded by changing the viscosity of solvent to raise the value of τ R . To further explore this concept, we prepared a series of samples of α-spectrin SH3 domain in solvent with increasing concentration of glycerol. In addition to the conventional 15N labeling, the protein was labeled in the Val, Leu methyl positions (13CHD2 on a deuterated background). The collected relaxation data were used in asymmetric fashion: backbone 15N relaxation rates were used to determine τ R across the series of samples, while methyl 13C data were used to probe local dynamics (side-chain motions). In interpreting the results, it has been initially suggested that addition of glycerol leads only to increases in τ R , whereas local motional parameters remain unchanged. Thus the data from multiple samples can be analyzed jointly, with τ R playing the role of experimentally controlled variable. Based on this concept, the extended model-free model was constructed with the intent to capture the effect of ns time-scale rotameric jumps in valine and leucine side chains. Using this model, we made a positive identification of nanosecond dynamics in Val-23 where ns motions were already observed earlier. In several other cases, however, only tentative identification was possible. The lack of definitive results was due to the approximate character of the model—contrary to what has been assumed, addition of glycerol led to a gradual ‘stiffening’ of the protein. This and other observations also shed light on the interaction of the protein with glycerol, which is one of the naturally occurring osmoprotectants. In particular, it has been found that the overall protein tumbling is controlled by the bulk solvent, and not by a thin solvation layer which contains a higher proportion of water.

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Notes

  1. An additional complication arises from the fact that CspB is thoroughly affected by ms time scale exchange arising from the folding–unfolding equilibrium that exists in this marginally stable protein. Addition of ethylene glycol shifts this equilibrium toward the folded form and thus alters the R ex rates.

  2. In order to extrapolate the results toward the limit of zero glycerol concentration the authors relied on the data from translational diffusion measurements. It should be pointed out that these measurements can be technically demanding; their accuracy is typically much lower than that of the standard τ R determination (Price 1998). In addition, a simple relationship between the translational and rotational diffusion coefficients, in principle, can be violated as a result of the solvent partition (i.e. preferential hydration of the protein surface).

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Acknowledgments

We are grateful to Bernd Reif, Veniamin Chevelkov, and Vipin Agrawal for continuing fruitful collaboration on α-spc SH3. The research has been funded through the NSF grants MCB-044563 and CHE-0723718.

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  1. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907-2084, USA

    Jun Xu, Yi Xue & Nikolai R. Skrynnikov

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Xu, J., Xue, Y. & Skrynnikov, N.R. Detection of nanosecond time scale side-chain jumps in a protein dissolved in water/glycerol solvent. J Biomol NMR 45, 57–72 (2009). https://doi.org/10.1007/s10858-009-9336-9

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