Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography.

We present millisecond quantitative serial X-ray crystallography at 1.7 Å resolution demonstrating precise optical control of reversible population transfer from Trans-Cis and Cis-Trans photoisomerization of a reversibly switchable fluorescent protein, rsKiiro. Quantitative results from the analysis of electron density differences, extrapolated structure factors, and occupancy refinements are shown to correspond to optical measurements of photoinduced population transfer and have sensitivity to a few percent in concentration differences. Millisecond time-resolved concentration differences are precisely and reversibly controlled through intense continuous wave laser illuminations at 405 and 473 nm for the Trans-to-Cis and Cis-to-Trans reactions, respectively, while the X-ray crystallographic measurement and laser illumination of the metastable Trans chromophore conformation causes partial thermally driven reconversion across a 91.5 kJ/mol thermal barrier from which a temperature jump between 112 and 128 K is extracted.

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 ß-barrel across the time window of our measurements.

Optical Screening and Classification of Drug Binding to Proteins in Human Blood Serum.

Protein-drug interactions in the human bloodstream are important factors in applications ranging from drug design, where protein binding influences efficacy and dose delivery, to biomedical diagnostics, where rapid, quantitative measurements could guide optimized treatment regimes. Current measurement approaches use multistep assays, which probe the protein-bound drug fraction indirectly and do not provide fundamental structural or dynamic information about the in vivo protein-drug interaction. We demonstrate that ultrafast 2D-IR spectroscopy can overcome these issues by providing a direct, label-free optical measurement of protein-drug binding in blood serum samples. Four commonly prescribed drugs, known to bind to human serum albumin (HSA), were added to pooled human serum at physiologically relevant concentrations. In each case, spectral changes to the amide I band of the serum sample were observed, consistent with binding to HSA, but were distinct for each of the four drugs. A machine-learning-based classification of the serum samples achieved a total cross-validation prediction accuracy of 92% when differentiating serum-only samples from those with a drug present. Identification on a per-drug basis achieved correct drug identification in 75% of cases. These unique spectroscopic signatures of the drug-protein interaction thus enable the detection and differentiation of drug containing samples and give structural insight into the binding process as well as quantitative information on protein-drug binding. Using currently available instrumentation, the 2D-IR data acquisition required just 1 min and 10 µL of serum per sample, and so these results pave the way to fast, specific, and quantitative measurements of protein-drug binding in vivo with potentially invaluable applications for the development of novel therapies and personalized medicine.

Open hardware microsecond dispersive transient absorption spectrometer for linear optical response.

An open hardware design and implementation for a transient absorption spectrometer are presented that has microsecond time resolution and measures full difference spectra in the visible spectral region from 380 to 750 nm. The instrument has been designed to allow transient absorption spectroscopy measurements of either low or high quantum yield processes by combining intense sub-microsecond excitation flashes using a xenon lamp together with stroboscopic non-actinic white light probing using LED sources driven under high pulsed current from a capacitor bank. The instrument is sensitive to resolve 0.15 mOD flash-induced differences within 1000 measurements at 20 Hz repetition rate using an inexpensive CCD sensor with 200 µm pixel dimension, 40 K electrons full well capacity and a dynamic range of 1800. The excitation flash has 230 ns pulse duration and the 2 mJ flash energy allows spectral filtering while retaining high power density with focussing to generate mOD signals in the 10-4-10-1 ΔOD range. We present the full electronics design and construction of the flash and probe sources, the optics as well as the timing electronics and CCD spectrometer operation and modification for internal signal referencing. The performance characterisation and example measurements are demonstrated using microsecond TAS of Congo red dye, as an example of a low quantum yield photoreaction at 2% with up to 78% of molecules excited. The instrument is fully open hardware and combines inexpensive selection of commercial components, optics and electronics and allows linear response measurements of photoinduced reactions for the purpose of accurate global analysis of chemical dynamics.

Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography.

Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue 'Measurement of ultrafast electronic and structural dynamics with X-rays'.

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.

XFEL Beamline Optical Instrumentation for Ultrafast Science.

Free electron lasers operating in the soft and hard X-ray regime provide capabilities for ultrafast science in many areas, including X-ray spectroscopy, diffractive imaging, solution and material scattering, and X-ray crystallography. Ultrafast time-resolved applications in the picosecond, femtosecond, and attosecond regimes are often possible using single-shot experimental configurations. Aside from X-ray pump and X-ray probe measurements, all other types of ultrafast experiments require the synchronized operation of pulsed laser excitation for resonant or nonresonant pumping. This Perspective focuses on the opportunities for the optical control of structural dynamics by applying techniques from nonlinear spectroscopy to ultrafast X-ray experiments. This typically requires the synthesis of two or more optical pulses with full control of pulse and interpulse parameters. To this end, full characterization of the femtosecond optical pulses is also highly desirable. It has recently been shown that two-color and two-pulse femtosecond excitation of fluorescent protein crystals allowed a Tannor-Rice coherent control experiment, performed under characterized conditions. Pulse shaping and the ability to synthesize multicolor and multipulse conditions are highly desirable and would enable XFEL facilities to offer capabilities for structural dynamics. This Perspective will give a summary of examples of the types of experiments that could be achieved, and it will additionally summarize the laser, pulse shaping, and characterization that would be recommended as standard equipment for time-resolved XFEL beamlines, with an emphasis on ultrafast time-resolved serial femtosecond crystallography.

The Carbonyl Group in ß2 of the Carotenoid Tunes the Photocycle Kinetics in Orange Carotenoid Protein.

Adaptation to rapid environmental changes is crucial for maintaining optimal photosynthetic efficiency and is ultimately key to the survival of all photosynthetic organisms. Like most of them, cyanobacteria protect their photosynthetic apparatus against rapidly increasing light intensities by nonphotochemical quenching (NPQ). In cyanobacteria, NPQ is controlled by Orange Carotenoid Protein (OCP) photocycle. OCP is the only known photoreceptor that uses carotenoid for its light activation. How carotenoid drives and controls this unique photoactivation process is still unknown. However, understanding and potentially controlling the OCP photocycle may open up new possibilities for improving photosynthetic biomass. Here we investigate the effect of the carbonyl group in the ß2 ring of the carotenoid on the OCP photocycle. We report microsecond to minute OCP light activation kinetics and Arrhenius plots of the two OCP forms: Canthaxanthin-bound OCP (OCPCAN) and echinenone-bound OCP (OCPECH). The difference between the two carotenoids is the presence of a carbonyl group in the ß2-ring located in the N-terminal domain of the protein. A combination of temperature-dependent spectroscopy, flash photolysis, and pump-probe transient absorption allows us to report the previously unresolved OCP intermediate associated primarily with the absorption bleach (OCPB). OCPB dominates the photokinetics in the µs to subms time range for OCPCAN and in the µs to ms range for OCPECH. We show that in OCPCAN the OCP photocycle steps are always faster than in OCPECH: from 2 to almost 20 times depending on the step. These results suggest that the presence of the carbonyl group in the ß2-ring of the carotenoid accelerates the OCP photocycle.

Laser induced temperature-jump time resolved IR spectroscopy of zeolites.

Combining pulsed laser heating and time-resolved infrared (TR-IR) absorption spectroscopy provides a means of initiating and studying thermally activated chemical reactions and diffusion processes in heterogeneous catalysts on timescales from nanoseconds to seconds. To this end, we investigated single pulse and burst laser heating in zeolite catalysts under realistic conditions using TR-IR spectroscopy. 1 ns, 70 µJ, 2.8 µm laser pulses from a Nd:YAG-pumped optical parametric oscillator were observed to induce temperature-jumps (T-jumps) in zeolite pellets in nanoseconds, with the sample cooling over 1-3 ms. By adopting a tightly focused beam geometry, T-jumps as large as 145 °C from the starting temperature were achieved, demonstrated through comparison of the TR-IR spectra with temperature dependent IR absorption spectra and three dimensional heat transfer modelling using realistic experimental parameters. The simulations provide a detailed understanding of the temperature distribution within the sample and its evolution over the cooling period, which we observe to be bi-exponential. These results provide foundations for determining the magnitude of a T-jump in a catalyst/adsorbate system from its absorption spectrum and physical properties, and for applying T-jump TR-IR spectroscopy to the study of reactive chemistry in heterogeneous catalysts.

Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE.

X-ray Free Electron Lasers (XFELs) allow the collection of high-quality serial femtosecond crystallography data. The next generation of megahertz superconducting FELs promises to drastically reduce data collection times, enabling the capture of more structures with higher signal-to-noise ratios and facilitating more complex experiments. Currently, gas dynamic virtual nozzles (GDVNs) stand as the sole delivery method capable of best utilizing the repetition rate of megahertz sources for crystallography. However, their substantial sample consumption renders their use impractical for many protein targets in serial crystallography experiments. Here, we present a novel application of a droplet-on-demand injection method, which allowed operation at 47 kHz at the European XFEL (EuXFEL) by tailoring a multi-droplet injection scheme for each macro-pulse. We demonstrate a collection rate of 150 000 indexed patterns per hour. We show that the performance and effective data collection rate are comparable to GDVN, with a sample consumption reduction of two orders of magnitude. We present lysozyme crystallographic data using the Large Pixel Detector at the femtosecond x-ray experiment endstation. Significant improvement of the crystallographic statistics was made by correcting for a systematic drift of the photon energy in the EuXFEL macro-pulse train, which was characterized from indexing the individual frames in the pulse train. This is the highest resolution protein structure collected and reported at the EuXFEL at 1.38 Å resolution.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy.

The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm-1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.

Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein.

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.