Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements

A radical-triggered reaction mechanism of the green-to-red photoconversion of EosFP

Reaction intermediates in the green-to-red photoconversion of the photochromic fluorescent protein EosFP have been observed using high-intensity continuous blue illumination. An intermediate was identified through light induced accumulation that continues to convert the green form in subsequent darkness, putatively containing a tyrosyl radical, albeit with anomalously shifted features in both electronic and FTIR spectra. Lowering the pH to 5.5 significantly delays the decay of this tyrosyl intermediate, which is accompanied by Stark-shifted features in the electronic spectra of reactants and products. Vibrational mode assignments for the high frequency and fingerprint FTIR spectral regions of reaction intermediates support a proposed sequence of events where the newly formed Cα=Cβ ethylenic bond precedes modifications on the His62 imidazole ring and confirms a C=O(NH2) product group on Phe61. We propose a reaction mechanism that involves tyrosyl generation via singlet excited state mediated oxidation which subsequently triggers the covalent reactions by oxidation of the green chromophore

The evolution and evolvability of photosystem II

Photosystem II is the water-oxidizing and O2 -evolving enzyme of photosynthesis. How and when this remarkable enzyme arose are fundamental questions in the history of life that have remained difficult to answer. Here, recent advances in our understanding of the origin and evolution of photosystem II are reviewed and discussed in detail. The evolution of photosystem II indicates that water oxidation originated early in the history of life, long before the diversification of cyanobacteria and other major groups of prokaryotes, challenging and transforming current paradigms on the evolution of photosynthesis. We show that photosystem II has remained virtually unchanged for well over three billion years, and yet the nonstop duplication process of the D1 subunit of photosystem II, which controls photochemistry and catalysis, has enabled the enzyme to become adaptable to variable environmental conditions, and even to innovate enzymatic functions beyond water oxidation. It is suggested that this evolvability can be exploited to develop novel light-powered enzymes with the capacity to carry out complex multi-step oxidative transformations for sustainable biocatalysis

Coincidence timing of femtosecond optical pulses in an X-ray free electron laser

Femtosecond resolution pump-probe experiments are now routinely carried out at X-ray Free Electron Lasers, enabled by the development of cross-correlation “time-tools” which correct the picosecond-level jitter between the optical and X-ray pulses. These tools provide very accurate, <10 fs, measurement of the relative arrival time, but do not provide a measure of the absolute coincidence time in the interaction. Cross-correlation experiments using transient reflectivity in a crystal are commonly used for this purpose, and to date no quantitative analysis of the accuracy or stability of absolute coincidence time determination has been performed. We have performed a quantitative analysis of coincidence timing at the SACLA facility through a cross-correlation of 100 6 10 fs, 400 nm optical pulses with 7 fs, 10.5 keV X-ray pulses via transient reflectivity in a cerium-doped yttrium aluminum garnet crystal. We have modelled and fit the transient reflectivity, which required a convolution with a 226 6 12 fs uncertainty that was believed to be dominated by X-ray and laser intensity fluctuations, or assuming an extinction depth of 13.3 lm greater than the literature value of 66.7 lm. Despite this, we are able to determine the absolute coincidence time to an accuracy of 30 fs. We discuss the physical contributions to the uncertainty of coincidence time determination, which may include an uncharacterised offset delay in the development of transient reflectivity, including cascading Auger decays, secondary ionisation and cooling processes. Additionally, we present measurements of the intrinsic short-term and long-term drifts between the X-rays and the optical laser timing from time-tool analysis, which is dominated by a thermal expansion of the 25 m optical path between tool and the interaction region, seen to be 60 fs over a period of 5 h

X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature.

The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form

Cds1 Controls the Release of Cdc14-like Phosphatase Flp1 from the Nucleolus to Drive Full Activation of the Checkpoint Response to Replication Stress in Fission Yeast

The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p. On replication stress, ataxia-telangiectasia mutated/ATM/Rad3-related kinase Rad3p activates Cds1p, which phosphorylates key proteins ensuring the stability of stalled DNA replication forks. Here, we show that replication stress induces changes in the subcellular localization of Flp1p in a checkpoint-dependent manner. Active Cds1p checkpoint kinase is required to release Flp1p into the nucleus. Consistently, a Flp1p mutant (flp1-9A) lacking all potential Cds1p phosphorylation sites fails to relocate in response to replication blocks and, similarly to cells lacking flp1 (Δflp1), presents defects in checkpoint response to replication stress. Δflp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, Δflp1 and flp1-9A have an increased percentage of Rad22p-recombination foci during HU treatment. Together, our data show that by releasing Flp1p into the nucleus Cds1p checkpoint kinase modulates its own full activation during replication stress