Membrane Protein Dynamics Revealed by X-Ray Scattering with a Femtosecond Free-Electron Laser

Abstract

The recent development of ultrafast X-ray free-election lasers (XFELs) gives a new avenue to experimentally investigate protein dynamics in the picosecond and femtosecond time regimes. The XFEL beam of the Linac Coherent Light Source (LCLS) has a very high peak X-ray brightness, which is key for probing elusive ultra-fast structural changes during the biological function of protein molecules. With XFEL probes, 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 technology in pump-probe, TR-SWAXS experiments to structurally interrogate the conformational changes of membrane proteins, with an emphasis on G-protein-coupled receptors. Rhodopsin in detergent micelles was delivered to the X-ray beam using micro-jet technology. Time-resolved data were generated by recording X-ray scattering following the pump-laser illumination (light) as well as X-ray scattering without pump-laser illumination (dark). The 2D X-ray scattering patterns were radially integrated to generate 1D scattering profiles versus momentum transfer vector (Q). On-the-fly data analysis using the OnDA software package revealed the light-triggered “protein quake” of rhodopsin during early stages of its activation (within 10 ps). Double-difference profiles of rhodopsin-opsin solution scattering from −200 fs to 100 ps emphasized the structural effects of photoactivation. The signal from retinal after light excitation propagates as a shock wave throughout the protein and into the solvent. Theoretical molecular dynamics (MD) simulations provide a molecular interpretation and show that the calculated radius of gyration (Rg) of rhodopsin increases upon light excitation. Pump-probe experiments of photoactive proteins using an XFEL bring structural biology to a new level that will enable high-resolution structural movies 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.