TY - JOUR

T1 - Time-resolved functional expansion of visual rhodopsin detected by x-ray scattering

AU - Menon, C. S. K.

AU - Karpos, K.

AU - Grant, Thomas D.

AU - Struts, A.V.

AU - Fried, Steven D. E.

AU - Alvarez, Roberto C.

AU - Perera, Suchithranga M. D. C.

AU - Kosheleva, I. V.

AU - Grossfield, Alan

AU - Fromme, Petra

AU - Kirian, Richard A.

AU - Brown, M.F.

PY - 2022/2/1

Y1 - 2022/2/1

N2 - Visual rhodopsin is an excellent model system to understand spatio-temporal dynamics of class A G-protein-coupled receptors (GPCRs). We aim to understand the mechanistic details of how the local changes in the retinal cofactor propagate to become large scale conformational changes leading to visual signaling. Our hypothesis was that a volumetric expansion occurs in the light-activation process [1]. Here we show the first time-resolved X-ray scattering-based detection of the 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 at the BioCARS synchrotron beamline (Advanced Photon Source). Difference scattering signals in both SAXS and WAXS regions were measured for rhodopsin pumped with actinic green (527-nm) ns pulses and probed with X-rays at 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 confirmed the capture of structural information from the retinal chromophore excitation. Optical (pump) laser power titrations 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 TR-SAXS region. We propose that 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. Our preliminary analysis shows time-resolved increases in the radius of gyration and the volume of the light-activated state versus the inactive dark state which are important for effector proteins to function in visual signaling.

AB - Visual rhodopsin is an excellent model system to understand spatio-temporal dynamics of class A G-protein-coupled receptors (GPCRs). We aim to understand the mechanistic details of how the local changes in the retinal cofactor propagate to become large scale conformational changes leading to visual signaling. Our hypothesis was that a volumetric expansion occurs in the light-activation process [1]. Here we show the first time-resolved X-ray scattering-based detection of the 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 at the BioCARS synchrotron beamline (Advanced Photon Source). Difference scattering signals in both SAXS and WAXS regions were measured for rhodopsin pumped with actinic green (527-nm) ns pulses and probed with X-rays at 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 confirmed the capture of structural information from the retinal chromophore excitation. Optical (pump) laser power titrations 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 TR-SAXS region. We propose that 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. Our preliminary analysis shows time-resolved increases in the radius of gyration and the volume of the light-activated state versus the inactive dark state which are important for effector proteins to function in visual signaling.

UR - https://www.mendeley.com/catalogue/d78f60c2-4fc6-3ea4-b53e-07e99743b094/

U2 - 10.1016/j.bpj.2021.11.1313

DO - 10.1016/j.bpj.2021.11.1313

M3 - Meeting Abstract

VL - 121

SP - 285A-286A

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 3 S1

Y2 - 19 February 2022 through 23 February 2022

ER -