TY - JOUR

T1 - Functional dynamics of G-protein-coupled receptor shown by femtosecond X-ray scattering

AU - Brown, M.F.

AU - Grant, Thomas D.

AU - Perera, Suchithranga M. D. C.

AU - Salas-Estrada, Leslie A.

AU - Struts, A.V.

AU - Karpos, K.

AU - Chawla, Udeep

AU - Fried, Steven D. E.

AU - Menon, C. S. K.

AU - Weerasinghe, Nipuna

AU - Meza, Domingo

AU - Mendez, Derek

AU - Grossfield, Alan

AU - Fromme, Petra

AU - Kirian, Richard A.

PY - 2022/2/1

Y1 - 2022/2/1

N2 - 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. For G-protein-coupled receptors (GPCRs) like rhodopsin, an important question is how the rapid local dynamics of the ligand initiate the functional protein transitions. However, structural evidence is lacking because until now the extreme reaction speed has precluded direct X-ray diffraction approaches. Here we show how solution X-ray scattering combined with molecular simulations informs the functional dynamics of membrane proteins in ways that could previously only be imagined. To follow the protein structural changes due to light absorption by retinal, we conducted time-resolved pump-probe small-angle X-ray scattering (TR-SAXS) studies of detergent-solubilized rhodopsin. Femtosecond solution X-ray scattering using an XFEL detected the protein structural change in the absence of a crystal lattice, and allowed the first direct detection of cis-trans isomerization of retinal bound to visual rhodopsin as it occurred in real time. We discovered a significant difference scattering signal within the first few time points immediately following the pump laser triggering event. Our analysis of the difference scattering profiles showed two dominant states within first 10 picoseconds due to the photorhodopsin and bathorhodopsin intermediates. Molecular dynamics (MD) simulations furthermore allowed interpretation of the structural motions corresponding to the cis-trans isomerization of retinal within the protein binding pocket. Upon light excitation the localized retinal isomerization initiates ultrafast global movements of the transmembrane helices that propagate at the speed of sound throughout the entire protein. The overall volumetric expansion is driven by the global structural changes over longer time scales that trigger the visual signaling cascade. Our experiments directly reveal the initial structural steps of rhodopsin leading to the metarhodopsin equilibrium, G-protein activation, and subsequent visual signaling.

AB - 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. For G-protein-coupled receptors (GPCRs) like rhodopsin, an important question is how the rapid local dynamics of the ligand initiate the functional protein transitions. However, structural evidence is lacking because until now the extreme reaction speed has precluded direct X-ray diffraction approaches. Here we show how solution X-ray scattering combined with molecular simulations informs the functional dynamics of membrane proteins in ways that could previously only be imagined. To follow the protein structural changes due to light absorption by retinal, we conducted time-resolved pump-probe small-angle X-ray scattering (TR-SAXS) studies of detergent-solubilized rhodopsin. Femtosecond solution X-ray scattering using an XFEL detected the protein structural change in the absence of a crystal lattice, and allowed the first direct detection of cis-trans isomerization of retinal bound to visual rhodopsin as it occurred in real time. We discovered a significant difference scattering signal within the first few time points immediately following the pump laser triggering event. Our analysis of the difference scattering profiles showed two dominant states within first 10 picoseconds due to the photorhodopsin and bathorhodopsin intermediates. Molecular dynamics (MD) simulations furthermore allowed interpretation of the structural motions corresponding to the cis-trans isomerization of retinal within the protein binding pocket. Upon light excitation the localized retinal isomerization initiates ultrafast global movements of the transmembrane helices that propagate at the speed of sound throughout the entire protein. The overall volumetric expansion is driven by the global structural changes over longer time scales that trigger the visual signaling cascade. Our experiments directly reveal the initial structural steps of rhodopsin leading to the metarhodopsin equilibrium, G-protein activation, and subsequent visual signaling.

UR - https://www.mendeley.com/catalogue/0919f673-9537-3e15-a1c4-2f5f339a7942/

U2 - 10.1016/j.bpj.2021.11.1755

DO - 10.1016/j.bpj.2021.11.1755

M3 - Meeting Abstract

VL - 121

SP - 193A

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 3 S1

Y2 - 19 February 2022 through 23 February 2022

ER -