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Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme

Abstract

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.

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Fig. 1: Overview of temperature-jump SAXS/WAXS experiments.
Fig. 2: Temperature-jump data allow kinetic modelling of conformational dynamics.
Fig. 3: Time-resolved Guinier analysis.
Fig. 4: Eyring analysis of transition-state thermodynamics.
Fig. 5: Kinetic analysis of CypA variants.

Data availability

Scattering data are deposited at NIH Figshare (https://doi.org/10.35092/yhjc.9177143). Additional information and files are available from the corresponding author upon reasonable request.

Code availability

All Python scripts used for analysis of integrated X-ray scattering curves are publicly available from GitHub (https://github.com/fraser-lab/solution_scattering). A code release checkpoint containing the exact scripts used in this work is available via Zenodo (https://doi.org/10.5281/zenodo.3355707).

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Acknowledgements

We thank R. Ranganathan, J. Holton, G. Hura and D. Elnatan for helpful discussions, and the staff at the BioCARS beamline at the Advanced Photon Source (I. Kosheleva, R. Henning, A. DiChiara and V. Srajer) for assistance. This work was supported by the NSF (STC-1231306), the NIH (GM123159 and GM124149), a Packard Fellowship from the David and Lucile Packard Foundation, the UC Office of the President Laboratory Fees Research Program LFR-17-476732 (to J.S.F.), the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (to P.A.) and a Ruth L. Kirschstein National Research Service Award (F32 HL129989 to M.C.T.). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract number DE-AC02-06CH11357. Use of the BioCARS Sector 14 was also supported by the NIH National Institute of General Medical Sciences (grant R24GM111072). The time-resolved setup at Sector 14 was funded in part through a collaboration with P.A. (NIH/NIDDK).

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M.C.T., P.A. and J.S.F. conceived and designed the experiments. M.C.T., B.A.B., A.M.W., H.S.C., F.S., D.M.C.S. and P.A. performed the experiments. M.C.T., B.A.B. and A.M.W. analysed the data. M.C.T., B.A.B., A.M.W., H.S.C., F.S. and P.A. contributed materials/analysis tools. M.C.T. and J.S.F. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Philip Anfinrud or James S. Fraser.

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Thompson, M.C., Barad, B.A., Wolff, A.M. et al. Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme. Nat. Chem. 11, 1058–1066 (2019). https://doi.org/10.1038/s41557-019-0329-3

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