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. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. 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

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

2D-IR VIBRATIONAL ECHO SPECTROSCOPY OF CONDUCTING POLYMERS

Author Institution: University of Minnesota, Department of Chemistry, 207 Pleasant St SE, Minneapolis MN 554552D-IR vibrational echo spectroscopy was used to examine the structural dynamics of various conducting polymers. A metal carbonyl was embedded in the polymer samples to serve as a global reporter of dynamics in these polymer thin films. Data taken on samples with metal carbonyl reporter species dissolved in NMP and embedded in polymer thin films will be presented. From these data, we are able to describe the time scales of structural motions present in these materials

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

High-resolution wavefront sensing of multi-spectral high-harmonic generation sources using ptychography

We perform high-resolution multi-spectral wavefront sensing on extreme ultraviolet sources produced by high-harmonic generation processes. Using ptychography, we show spectrally resolved complex-valued beam reconstructions for eight harmonics simultaneously, with a spatial resolution of 1 µm

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

WOS:000455561800070International audienceThe 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 beta 1-ring in picoseconds occurs at a low yield of \textless1%, whereby the beta 1-ring retains a trans configuration with respect to the conjugated pi-electron chain. This event initiates structural changes at the N-terminal domain in 1 mu s, which allow the carotenoid to translocate into the N-terminal domain in 10 mu s. We identified infrared signatures of helical elements that dock on the C-terminal domain beta-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 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. 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 β-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