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.

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.

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.

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.

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 (λ = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme.

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.

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 OCP; blue light illumination results in the red active state, known as OCP. The OCP 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.

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 β-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.

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.

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.

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.

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.

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.