Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs.

Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes.

3D-printed sheet jet for stable megahertz liquid sample delivery at X-ray free-electron lasers.

X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60 nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13 MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

The molecular pH-response mechanism of the plant light-stress sensor PsbS.

Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses.

NeoR, a near-infrared absorbing rhodopsin.

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λmax = 690 nm) highly fluorescent state (QF = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein.

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.

Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy.

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a ß-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 µs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 µs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the ß-sheet, and an additional helical element. The second phase occurs in approximately 240 µs. The final spectrum at 500 µs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Far-Red Emission of mPlum Fluorescent Protein Results from Excited-State Interconversion between Chromophore Hydrogen-Bonding States.

Fluorescent proteins with large Stokes shifted emission beyond 600 nm are actively sought for live-cell imaging applications. The mechanism of excited-state relaxation leading to the Stokes shift in the mPlum fluorescent protein, which emits at a peak wavelength of 650 nm, has been previously investigated by both ultrafast spectroscopy and theoretical methods. Here, we report that femtosecond time-resolved area-normalized emission spectra of mPlum show a clear isoemissive point. This feature can only result from a system with two emitting states, rather than a system that undergoes a continuous spectral red shift, for example, as expected from typical solvation. Global analysis of the femtosecond time-resolved fluorescence spectra reveals time constants associated with chromophore relaxation, excited-state population transfer, and an excited-state lifetime of the final state. The observations confirm the findings of recent quantum chemical calculations on mPlum.

Femtosecond Measurements Of Size-Dependent Spin Crossover In Fe(II)(pyz)Pt(CN)4 Nanocrystals.

We report a femtosecond time-resolved spectroscopic study of size-dependent dynamics in nanocrystals (NCs) of Fe(pyz)Pt(CN)4. We observe that smaller NCs (123 or 78 nm cross section and <25 nm thickness) exhibit signatures of spin crossover (SCO) with time constants of ∼5-10 ps whereas larger NCs with 375 nm cross section and 43 nm thickness exhibit a weaker SCO signature accompanied by strong spectral shifting on a ∼20 ps time scale. For the small NCs, the fast dynamics appear to result from thermal promotion of residual low-spin states to high-spin states following nonradiative decay, and the size dependence is postulated to arise from differing high-spin vs low-spin fractions in domains residing in strained surface regions. The SCO is less efficient in larger NCs owing to their larger size and hence lower residual LS/HS fractions. Our results suggest that size-dependent dynamics can be controlled by tuning surface energy in NCs with dimensions below ∼25 nm for use in energy harvesting, spin switching, and other applications.

Excited state electronic landscape of mPlum revealed by two-dimensional double quantum coherence spectroscopy.

Red fluorescent proteins (RFPs) are widely used probes for monitoring subcellular processes with extremely high spatial and temporal precision. In this work, we employed spectrally resolved transient absorption (SRTA) and two-dimensional double quantum coherence (2D2Q) spectroscopy to investigate the excited state electronic structure of mPlum, a well-known RFP. The SRTA spectra reveal the presence of excited state absorption features at both the low- and high-energy sides of the dominant ground state bleach contribution. The 2D2Q spectra measured at several excitation wavelengths reveal a peak pattern consistent with the presence of more than three electronic states (i.e., ground, excited, and doubly excited). Numerical modeling of this response suggests that the features are consistent with a 1-1-2 electronic structure. The two closely spaced (∼1500 cm(-1)) levels in the double quantum manifold appear at opposite anharmonicities relative to twice the energy of the lowest energy transition. These observations explain the excited state absorption contributions observed in spectrally resolved transient grating and transient absorption measurements and demonstrate the utility of multidimensional spectroscopy in unraveling congested spectra relative to conventional one-dimensional methods.

Hydrogen bond flexibility correlates with Stokes shift in mPlum variants.

Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins.

Infrared spectroscopic signatures of phase segregation in P3HT-porphyrin blends.

Phase segregation of 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphine ruthenium(II)carbonyl (RuOEP) and regioregular poly(3-hexylthiophene-2,5-diyl) (P3HT) in thin films is investigated with infrared and UV-visible spectroscopies as well as transmission electron microscopy (TEM). The Fourier transform infrared (FTIR) spectrum of the ruthenium-bound CO symmetric stretching mode exhibits significant changes as these films are annealed in solvent vapors. The development of multiple inhomogeneously broadened microenvironments is observed, and UV-visible spectra and TEM support a model of homogeneous porphyrin distribution throughout the P3HT films that gradually becomes more heterogeneous as the P3HT and RuOEP molecules phase segregate. A complete model for the phase segregation process experienced by the embedded RuOEP is proposed to explain the collective experimental observations.