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Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster(6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cisretinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-transconformation2, 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 temperature3to determine how an isomerized twisted all-transretinal 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.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastusrhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivopotential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivowith cellular precision and second-level resolution. These nanomolar, in vivocapable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivophotopharmacology one step closer to general realization.