Abstract
Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-013-9787-x) contains supplementary material, which is available to authorized users.
Highlights
The three-dimensional structure that a protein spontaneously adopts in its environment is dictated by a subtle balance of numerous interactions, which are all individually weak
We show that 15N relaxation data are generally insufficient to correctly describe amide backbone dynamics, even when different types of relaxation rate constants are measured at multiple static magnetic field strengths
We estimate that with these careful calibrations and corrections, the systematic error of the obtained dipolar couplings can be below 1% at most, as suggested by the close correspondence of solution- and solid-state order parameters
Summary
The three-dimensional structure that a protein spontaneously adopts in its environment is dictated by a subtle balance of numerous interactions, which are all individually weak. Different approaches have been proposed in recent studies of protein dynamics, as to which type of the above data should be used for determination of motional parameters, as well as to how these experimental data should be acquired (Chevelkov et al 2009a, b; Lewandowski et al 2011; Schanda et al 2010; Yang et al 2009), and even whether they should be interpreted in terms of local or global motion (Lewandowski et al 2010a) In this manuscript, we systematically investigate ways to determine backbone dynamics in proteins using various longitudinal and transverse 15N relaxation rates, as well as 1H–15N dipolar coupling measurements. Of up to 7 data points per residue), we investigate backbone mobility in microcrystalline ubiquitin, and compare the results to solution-state NMR data
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