The development of ultrafast X-ray free-electron lasers (XFELs) gives a new avenue to experimentally investigate protein dynamics in the picosecond and femtosecond time regimes. The XFEL beam of the LCLS has a very high peak X-ray brightness, which is key for probing elusive ultrafast structural changes during the biological function of protein molecules. With XFELs, femtosecond time-resolved small- and wide-angle X-ray scattering (TR-SWAXS) experiments afford unprecedented opportunities in membrane biophysics. Here we describe the application of XFEL pump-probe TR-SWAXS experiments to structurally interrogate the conformational changes of membrane proteins, with an emphasis on G-protein coupled receptors. Visual rhodopsin in detergent micelles was delivered to the X-ray beam using micro-jet technology, and pump-probe TR-SWAXS data were collected using a femtosecond 527-nm laser pulse to trigger the 11-<i>cis</i> to all-<i>trans</i> isomerization of retinal. The 2D X-ray scattering patterns were radially integrated to generate 1D TR-SWAXS profiles to probe time scales from –200 fs to 10 ps after photoactivation. We combined particle size analysis of the scattering profiles with independent, extensive theoretical molecular dynamics (MD) simulations to provide a molecular interpretation of the XFEL data. Results from both the experimental data and theoretical simulations show that local structural changes including the isomerization of retinal occur within hundreds of femtoseconds after light excitation, followed by global structural changes including the expansion of the protein, which propagate outward throughout the protein and into the solvent within several picoseconds. Our results show that ultrafast pump-probe TR-SWAXS experiments at XFELs combined with MD simulations of photoactive proteins is a powerful method for probing the structural biology of biomolecules in action. Establishing whether there is a single activation step or an energy-landscape will be significant to understanding the functional GPCR activation mechanisms.

Ultrafast membrane protein dynamics revealed by X-ray scattering with a free-electron laser

ACS Spring Meeting 2021

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