Ultrafast structural changes direct the first molecular events of vision

Abstract

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Conformational flexibility of β‐arrestins – How these scaffolding proteins guide and transform the functionality of GPCRs

“…[36][37][38][39] Interestingly, also X-ray crystallography is constantly developed further, as we can now appreciate how the use of free-electron lasers is able to bridge the gap between the elucidation of a static structures by capturing conformational changes of proteins within microcrystals at a femtosecond resolution. [40][41][42][43] Nevertheless, the throughput of cryo-EM, as well as X-ray crystallography structure determination, specifically regarding membrane proteins, is very low and associated with large experimental effort and expenses.…”

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism

“…As a volume-conserving alternative to the OBF for isomerization in bathorhodopsin, they proposed the HT mechanism, where both τ and the neighboring bond ϕ rotate simultaneously (Figure bottom). Since it has become generally accepted that photoisomerization in rhodopsins occurs through a bicycle-pedal mechanism, in which the cis conformation is propagated along the chromophore by a concerted rotation about parallel pairs of double bonds and not through a HT. − A recent time-resolved crystallographic study has in fact obtained structural evidence for an aborted bicycle-pedal mechanism in a bovine rhodopsin starting at 1 ps. , Nonetheless, crystallographic data supporting the presence of two volume-conserving isomerization pathways, including the HT, were obtained for nanosecond intermediates of the photoactive yellow protein (PYP) photocycle …”

“…19 bicycle-pedal mechanism in a bovine rhodopsin starting at 1 ps. 22,23 Nonetheless, crystallographic data supporting the presence of two volume-conserving isomerization pathways, including the HT, were obtained for nanosecond intermediates of the photoactive yellow protein (PYP) photocycle. 24 Conclusive experimental evidence or consensus for whether GFP-like chromophores that are functionally embedded in proteins undergo OBF or HT as the primary photoactivation pathway has not yet been obtained.…”

Introduction to the virtual thematic issue on room-temperature biological crystallography

“…Also, temperature-dependent differences in protein-ligand interactions including unique binding poses and new binding sites (Skaist Mehlman et al, 2023) make RT crystallographic studies very relevant for a more complete understanding of protein-ligand interactions that might be important for drug discovery (Fischer et al, 2014;Fischer et al, 2015). Importantly, RT studies on microcrystals coupled with suitable mixing and/or activation methods can also allow the visualization of reaction intermediates (Olmos et al, 2018;Butryn et al, 2021;Gruhl et al, 2023).…”