A guide to time‐resolved structural analysis of light‐activated proteins

Abstract

Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Lightactivated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light-absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enable the desired chemical and/or biological output response. Time-resolved serial-femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.

Detailed analysis of distorted retinal and its interaction with surrounding residues in the K intermediate of bacteriorhodopsin

“…There is no dispute that TR-SFX makes a great contribution to the study of protein conformational changes. However, the intense light pulses of pump lasers have been used as a trigger in the determination of reaction intermediates in TR-SFX 39 . It has been pointed out that the contribution of multiphoton processes due to high photon densities can be problematic in such experiments 40 .…”

Understanding Cysteine Chemistry Using Conventional and Serial X-ray Protein Crystallography

“…Each can be used to initiate catalysis at a defined time across the many molecules in the crystal lattice followed by monitoring the structures of the protein at defined timepoints after catalysis begins. Systems that are sensitive to light are ideal candidates for photoinitiation followed by time-resolved crystallography and include photosystem II [ 12 , 67 , 76 ], fatty acid photodecarboxylase [ 11 ], and photolyase [ 77 ] as well as a number of photoproteins that are not enzymes [ 78 ]. However, enzymes whose reactions are initiated by light are rare.…”

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

“…Although a variety of proteins are light sensitive and amenable to light-driven time-resolved studies [ 40 ], mixing crystals with substrate provides a more widely applicable means of triggering catalysis [ 19 , ∗41 , 42 ]. The achievable time resolution is determined by diffusion and the size of the crystals should be carefully assessed with reference to the time points being probed [ 43 ].…”