High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore2-14. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern15 whether this experimental approach16 allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions17. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics15) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments16 such that biologically relevant insight emerges.

Influence of pump laser fluence on ultrafast structural changes in myoglobin

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design. 

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers

Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein–drug interactions, protein–protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.

Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions

Microtubule-targeting agents (MTAs) have demonstrated remarkable efficacy as antitumor, antifungal, antiparasitic, and herbicidal agents, finding applications in the clinical, veterinary, and agrochemical industry. Recent advances in tubulin and microtubule structural biology have provided powerful tools that pave the way for the rational design of innovative small-molecule MTAs for future basic and applied life science applications. In this mini-review, we present the current status of the tubulin and microtubule structural biology field, the recent impact it had on the discovery and rational design of MTAs, and exciting avenues for future MTA research. 

Structure-based discovery and rational design of microtubule-targeting agents

Antimitotic agents such as the clinically approved vinca alkaloids, taxanes and epothilone can arrest cell growth during interphase and are therefore among the most important drugs available for treating cancer. These agents suppress microtubule dynamics and thus interfere with intracellular transport, inhibit cell proliferation and promote cell death. Because these drugs target biological processes that are essential to all cells, they face an additional challenge when compared to most other drug classes. General toxicity can limit the applicable dose and therefore reduce therapeutic benefits. Photopharmacology aims to avoid these side-effects by introducing compounds that can be applied globally to cells in their inactive form, then be selectively induced to bioactivity in targeted cells or tissue during a defined time window. This review discusses photoswitchable analogues of antimitotic agents that have been developed by combining different photoswitchable motifs with microtubule-stabilizing or microtubule-destabilizing agents.

/pdf/photopharmacology-of-antimitotic-agents-lwmwwdpx.pdf

Photopharmacology of Antimitotic Agents

The dynamic interplay between proteins and their ligands is central to molecular biology, pharmacology, and drug development but is difficult to resolve experimentally. Using time-resolved serial crystallography at a synchrotron and X-ray laser, we studied the release of the photochemical affinity switch azo-Combretastatin A4 from the anti-cancer target tubulin. Thirteen logarithmically spaced temporal snapshots at near-atomic resolution are complemented by time-resolved spectroscopy and molecular dynamics simulations. They show how the photoinduced cis to trans isomerization of the azobenzene bond stretches the ligand in the picosecond to nanosecond range, followed by stepwise opening of a gating loop within microseconds, and completion of the unbinding reaction within milliseconds. Ligand unbinding is accompanied by collapse of the binding pocket and global tubulin-backbone rearrangements. Our results have implications for the molecular basis of photopharmacology, the mechanism of action of anti-tubulin drugs and provide a general experimental framework to study protein-ligand interaction dynamics.Time-resolved crystallography reveals structural changes upon release of a photoswitchable inhibitor of microtubule dynamics.

Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

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Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time