RT Journal Article
SR Electronic
T1 Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 476432
DO 10.1101/476432
A1 Michael C. Thompson
A1 Benjamin A. Barad
A1 Alexander M. Wolff
A1 Hyun Sun Cho
A1 Friedrich Schotte
A1 Daniel M.C. Schwarz
A1 Philip Anfinrud
A1 James S. Fraser
YR 2018
UL http://biorxiv.org/content/early/2018/12/04/476432.abstract
AB Correlated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the μs-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.