Visual rhodopsin forms an excellent archetype for studying spatial and temporal protein dynamics in G- protein-coupled receptors (GPCR) of the human genome. We have proposed a volumetric expansion in rhodopsin following its photoactivation; however, a time-resolved description is lacking. Here we show the first time-resolved X-ray scattering-based detection of such an expansion of rhodopsin leading to the signaling state. We conducted time-resolved small-angle and wide-angle scattering (TR-SAXS and TR-WAXS, respectively) of rhodopsin solubilized in CHAPS detergent micelles at the BioCARS synchrotron (Advanced Photon Source). Difference scattering signals in both the low and high q=(4π/λ)sinθ regions were measured for rhodopsin pumped with actinic green (527 nm) ns pulses, and pump X-ray-probe time delays between 10 ns to 128 ms. Our controls were the retinal-free apoprotein opsin pumped with either green (527 nm) or red (650 nm) light, and the rhodopsin holoprotein pumped with off-resonance red light (650 nm). The absence of difference scattering signals in both controls confirm the capture of structural information from the retinal chromophore excitation. Optical (pump) laser power titrations (14.8–170.4 MW/cm2) were conducted to understand the interplay between water heating found in the high-q region (2–2.5Å–1) (TR-WAXS) and the difference signal in the low-q (TR-SAXS) region. We found the water heating (at high laser power densities) cannot explain the fluctuations in the low-q difference signal even though they are correlated. We hypothesize the water heating corresponds to the sequential re-excitation and vibrational cooling of the bathorhodopsin (ns) intermediate after photoisomerization, with the excess energy ultimately dissipated to the bulk solvent. The Guinier-analysis of the difference profiles indicates the resultant water-induced swelling of the membrane protein giving rise to the light-activated signaling state in the visual process.

Time-resolved solution X-ray scattering reveals functional volumetric expansion of G-protein-coupled receptor

ACS Spring Meeting 2021

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