76 results found
Bressan G, van Thor JJ, 2021, Theory of two-dimensional spectroscopy with intense laser fields, The Journal of Chemical Physics, Vol: 154, Pages: 1-10, ISSN: 0021-9606
Two-dimensional vibrational and electronic spectroscopic observables of isotropically oriented molecular samples in solution are sensitive to laser field intensities and polarization. The third-order response function formalism predicts a signal that grows linearly with the field strength of each laser pulse, thus lacking a way of accounting for non-trivial intensity-dependent effects, such as saturation and finite bleaching. An analytical expression to describe the orientational part of the molecular response, which, in the weak-field limit, becomes equivalent to a four-point correlation function, is presented. Such an expression is evaluated for Liouville-space pathways accounting for diagonal and cross peaks for all-parallel and cross-polarized pulse sequences, in both the weak- and strong-field conditions, via truncation of a Taylor series expansion at different orders. The results obtained in the strong-field conditions suggest how a careful analysis of two-dimensional spectroscopic experimental data should include laser pulse intensity considerations when determining molecular internal coordinates.
Fare C, Yuan L, Cordon-Preciado V, et al., 2020, A radical-triggered reaction mechanism of the green-to-red photoconversion of EosFP, The Journal of Physical Chemistry B, Vol: 124, Pages: 7765-7778, ISSN: 1520-6106
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.
Zamzam N, Rakowski R, Kaucikas M, et al., 2020, Femtosecond visible transient absorption spectroscopy of chlorophyll f- containing Photosystem II, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 1-7, ISSN: 0027-8424
The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a. We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d. The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210–1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.
Fadini A, Reiche S, Nass K, et al., 2020, Applications and Limits of Time-to-Energy Mapping of Protein Crystal Diffraction Using Energy-Chirped Polychromatic XFEL Pulses, APPLIED SCIENCES-BASEL, Vol: 10
van Thor JJ, 2019, Advances and opportunities in ultrafast X-ray crystallography and ultrafast structural optical crystallography of nuclear and electronic protein dynamics, Structural Dynamics-US, Vol: 6, Pages: 1-17, ISSN: 2329-7778
Both nuclear and electronic dynamics contribute to protein function and need multiple and complementary techniques to reveal their ultrafast structural dynamics response. Real-space information obtained from the measurement of electron density dynamics by X-ray crystallography provides aspects of both, while the molecular physics of coherence parameters and frequency-frequency correlation needs spectroscopy methods. Ultrafast pump-probe applications of protein dynamics in crystals provide real-space information through direct X-ray crystallographic structure analysis or through structural optical crystallographic analysis. A discussion of methods of analysis using ultrafast macromolecular X-ray crystallography and ultrafast nonlinear structural optical crystallography is presented. The current and future high repetition rate capabilities provided by X-ray free electron lasers for ultrafast diffraction studies provide opportunities for optical control and optical selection of nuclear coherence which may develop to access higher frequency dynamics through improvements of sensitivity and time resolution to reveal coherence directly. Specific selection of electronic coherence requires optical probes, which can provide real-space structural information through photoselection of oriented samples and specifically in birefringent crystals. Ultrafast structural optical crystallography of photosynthetic energy transfer has been demonstrated, and the theory of two-dimensional structural optical crystallography has shown a method for accessing the structural selection of electronic coherence.
Zamzam N, van Thor JJ, 2019, Excited state frequencies of chlorophyll f and chlorophyll a and evaluation of displacement through franck-condon progression calculations, Molecules, Vol: 24, Pages: 1-15, ISSN: 1420-3049
We present ground and excited state frequency calculations of the recently discovered extremely red-shifted chlorophyll f. We discuss the experimentally available vibrational mode assignments of chlorophyll f and chlorophyll a which are characterised by particularly large downshifts of 131-keto mode in the excited state. The accuracy of excited state frequencies and their displacements are evaluated by the construction of Franck–Condon (FC) and Herzberg–Teller (HT) progressions at the CAM-B3LYP/6-31G(d) level. Results show that while CAM-B3LYP results are improved relative to B3LYP calculations, the displacements and downshifts of high-frequency modes are underestimated still, and that the progressions calculated for low temperature are dominated by low-frequency modes rather than fingerprint modes that are Resonant Raman active.
Hutchison CDM, van Thor J, 2019, Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography, Philosophical Transactions A: Mathematical, Physical and Engineering Sciences, Vol: 377, ISSN: 1364-503X
Ultrafast pump-probe X-ray crystallography has now been established at XFELs that operate at hard X-ray energies. We discussthe 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.
van Thor JJ, 2019, Coherent two-dimensional electronic and infrared crystallography, The Journal of Chemical Physics, Vol: 150, ISSN: 0021-9606
The two-dimensional electronic and infrared spectroscopy of oriented single crystals is sensitive to structure and point group symmetry. The third order response of crystals is generally different from measurements of isotropic solutions because each coherence path that contributes to the measured field scales to the ensemble average of the four-point correlation functions of the four field-dipole interactions involved in the respective Feynman paths. An analytical evaluation of 2D optical crystallography which depends on the crystal symmetry, laboratory orientation, and the orientation in the crystallographic frame is presented. Applying a symmetry operator in the basis of the allowed polarised radiation modes provides a method for evaluation of non-zero fourth rank tensor elements alternative to direct inspection methods. Uniaxial and biaxial systems are distinguished and the contributions to the rephasing and non-rephasing directions are evaluated for isolated and coupled oscillators. By exploiting coordinate analysis, the extension of non-linear electronic and infrared crystallography for coupled oscillators demonstrates the structural, directional, and symmetry dependent selection of coherences to the four-wave mixing signal.
Zamzam N, Kaucikas M, Nurnberg D, et al., 2019, Femtosecond infrared spectroscopy of chlorophyll f-containing photosystem I, Physical Chemistry Chemical Physics, Vol: 21, Pages: 1224-1234, ISSN: 1463-9076
The recent discovery of extremely red-shifted chlorophyll f pigments in both photosystem I (PSI) and photosystem II has led to the conclusion that chlorophyll f plays a role not only in the energy transfer, but also in the charge separation processes [Nürnberg et al., Science, 2018, 360, 1210–1213]. We have employed ultrafast transient infrared absorption spectroscopy to study the contribution of far-red light absorbing chlorophyll f to energy transfer and charge separation processes in far-red light-grown PSI (FRL-PSI) from the cyanobacterium Chroococcidiopsis thermalis PCC 7203. We compare the kinetics and spectra of FRL-grown PSI excited at 670 nm and 740 nm wavelengths to those of white light-grown PSI (WL-PSI) obtained at 675 nm excitation. We report a fast decay of excited state features of chlorophyll a and complete energy transfer from chlorophyll a to chlorophyll f in FRL-PSI upon 670 nm excitation, as indicated by a frequency shift in a carbonyl absorption band occurring within a 1 ps timescale. While the WL-PSI measurements support the assignment of initial charge separation to A−1+˙A0−˙ [Di Donato et al., Biochemistry, 2011, 50, 480–490] from the kinetics of a distinct cation feature at 1710 cm−1, in the case of FRL-PSI, small features at 1715 cm−1 from the chlorophyll cation are present from sub-ps delays instead, supporting the replacement of the A−1 pigment with chlorophyll f. Comparisons of nanosecond spectra show that charge separation proceeds with 740 nm excitation, which selectively excites chlorophyll f, and modifications in specific carbonyl absorption bands assigned to P700+˙ minus P700 and A1−˙ minus A1 indicate dielectric differences of FRL-PSI compared to WL-PSI in one or both of the two electron transfer branches of FRL-PSI.
Sanchez-Gonzalez A, Johnson AS, Fitzpatrick A, et al., 2017, Coincidence timing of femtosecond optical pulses in an X-ray free electron laser, Journal of Applied Physics, Vol: 122, ISSN: 0021-8979
Femtosecond resolution pump-probe experiments are now routinely carried out at X-ray FreeElectron Lasers, enabled by the development of cross-correlation “time-tools” which correct thepicosecond-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 absolutecoincidence time in the interaction. Cross-correlation experiments using transient reflectivity in acrystal are commonly used for this purpose, and to date no quantitative analysis of the accuracy orstability of absolute coincidence time determination has been performed. We have performed aquantitative analysis of coincidence timing at the SACLA facility through a cross-correlation of100 6 10 fs, 400 nm optical pulses with 7 fs, 10.5 keV X-ray pulses via transient reflectivity in acerium-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 byX-ray and laser intensity fluctuations, or assuming an extinction depth of 13.3 lm greater than theliterature value of 66.7 lm. Despite this, we are able to determine the absolute coincidence time toan accuracy of 30 fs. We discuss the physical contributions to the uncertainty of coincidence timedetermination, which may include an uncharacterised offset delay in the development of transientreflectivity, including cascading Auger decays, secondary ionisation and cooling processes.Additionally, we present measurements of the intrinsic short-term and long-term drifts between theX-rays and the optical laser timing from time-tool analysis, which is dominated by a thermalexpansion of the 25 m optical path between tool and the interaction region, seen to be 60 fs overa period of 5 h.
Kacikas M, Renger T, van Thor J, 2017, Femtosecond Infrared Crystallography of Photosystem II core complexes: Watching exciton dynamics and charge separation in real space and time, Photosynthesis and Bioenergetics, Editors: Barber, Ruban, ISBN: 978-981-3230-29-3
Hutchison CDM, Cordon-Preciado V, Morgan RML, et al., 2017, X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature., International Journal of Molecular Sciences, Vol: 18, ISSN: 1422-0067
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.
Dorlhiac GF, Fare C, van Thor JJ, 2017, PyLDM - An open source package for lifetime density analysis of time-resolved spectroscopic data, Plos Computational Biology, Vol: 13, ISSN: 1553-7358
Ultrafast spectroscopy offers temporal resolution for probing processes in the femto- and picosecond regimes. This has allowed for investigation of energy and charge transfer in numerous photoactive compounds and complexes. However, analysis of the resultant data can be complicated, particularly in more complex biological systems, such as photosystems. Historically, the dual approach of global analysis and target modelling has been used to elucidate kinetic descriptions of the system, and the identity of transient species respectively. With regards to the former, the technique of lifetime density analysis (LDA) offers an appealing alternative. While global analysis approximates the data to the sum of a small number of exponential decays, typically on the order of 2-4, LDA uses a semi-continuous distribution of 100 lifetimes. This allows for the elucidation of lifetime distributions, which may be expected from investigation of complex systems with many chromophores, as opposed to averages. Furthermore, the inherent assumption of linear combinations of decays in global analysis means the technique is unable to describe dynamic motion, a process which is resolvable with LDA. The technique was introduced to the field of photosynthesis over a decade ago by the Holzwarth group. The analysis has been demonstrated to be an important tool to evaluate complex dynamics such as photosynthetic energy transfer, and complements traditional global and target analysis techniques. Although theory has been well described, no open source code has so far been available to perform lifetime density analysis. Therefore, we introduce a python (2.7) based package, PyLDM, to address this need. We furthermore provide a direct comparison of the capabilities of LDA with those of the more familiar global analysis, as well as providing a number of statistical techniques for dealing with the regularization of noisy data.
Hutchison CDM, van Thor JJ, 2017, Populations and coherence in femtosecond time resolved X-ray crystallography of the photoactive yellow protein, International Reviews in Physical Chemistry, Vol: 36, Pages: 117-143, ISSN: 0144-235X
Ultrafast X-ray crystallography of the photoactive yellow protein with femtosecond delays using an X-ray free electron laser has successfully probed the dynamics of an early Franck-Condon species. The femtosecond pump-probe application of protein crystallography represents a new experimental regime that provides an X-ray structural probe for coherent processes that were previously accessible primarily using ultrafast spectroscopy. We address how the optical regime of the visible pump, that is necessary to successfully resolve ultrafast structural differences, affects the motions that are measured using the technique. The sub-picosecond photochemical dynamics in PYP involves evolution of a mixture of electronic ground and excited state populations. Additional to photoisomerisation that is considered to proceed through activated barrier crossing, within the dephasing time structural motion include vibrational coherence arising from excited states, the ground state and a ground state intermediate under experimental conditions used for ultrafast crystallography. Intense optical pulses are required to convert population levels in PYP crystals that allow detection by X-ray crystallography, but the compromise currently needed for the optical bandwidth and power has consequences with regard to the contributions to the motions that are experimentally measured with femtosecond delays. We briefly review the ultrafast spectroscopy literature of the primary photoreactions of PYP and discuss relevant physical models taken from coherent control and femtosecond coherence spectroscopy literature that address both the population transfer as well as the vibrational coherences. We apply linear response theory, with the additional use of a high power approximation, of on-resonance impulsive vibrational coherence in the ground state and the non-impulsive coherence in the excited state and discuss experimental approaches to manipulate the coherence contributions. The results are generalis
Kaucikas M, Nurnberg D, Dorhliac G, et al., 2017, Femtosecond visible transient absorption spectroscopy ofChlorophyll f-containing Photosystem I, Biophysical Journal, Vol: 112, Pages: 234-249, ISSN: 1542-0086
Photosystem I (PSI) from Chroococcidiopsis thermalis PCC 7203 grown under far-red light (FRL; >725 nm) contains both chlorophyll a and a small proportion of chlorophyll f. Here, we investigated excitation energy transfer and charge separation using this FRL-grown form of PSI (FRL-PSI). We compared femtosecond transient visible absorption changes of normal, white-light (WL)-grown PSI (WL-PSI) with those of FRL-PSI using excitation at 670 nm, 700 nm, and (in the case of FRL-PSI) 740 nm. The possibility that chlorophyll f participates in energy transfer or charge separation is discussed on the basis of spectral assignments. With selective pumping of chlorophyll f at 740 nm, we observe a final ∼150 ps decay assigned to trapping by charge separation, and the amplitude of the resulting P700+•A1−• charge-separated state indicates that the yield is directly comparable to that of WL-PSI. The kinetics shows a rapid 2 ps time constant for almost complete transfer to chlorophyll f if chlorophyll a is pumped with a wavelength of 670 nm or 700 nm. Although the physical role of chlorophyll f is best supported as a low-energy radiative trap, the physical location should be close to or potentially within the charge-separating pigments to allow efficient transfer for charge separation on the 150 ps timescale. Target models can be developed that include a branching in the formation of the charge separation for either WL-PSI or FRL-PSI.
Kaucikas M, Maghlaoui K, barber J, et al., 2016, Ultrafast infrared observation of exciton equilibration from oriented single crystals of photosystem II, Nature Communications, Vol: 7, ISSN: 2041-1723
In oxygenic photosynthesis two photosystems work in series. Each of them contains a reaction center that is surrounded by light-harvesting antennae which absorb the light and transfer the excitation energy to the reaction center where electron transfer reactions are driven. Here, we report a critical test for two contrasting models of light harvesting by photosystem II cores, known as the trap-limited and the transfer-to-the trap limited model. Oriented single crystals of photosystem II core complexes of Synechococcus elongatus are excited by polarized visible light and the transient absorption is probed with polarized light in the infrared. The dichroic amplitudes resulting from photoselection are maintained on the 60 ps time scale that corresponds to the dominant energy transfer process providing compelling evidence for the transfer-to the–trap limitation of the overall light-harvesting process. This finding has functional implications for the quenching of excited states allowing plants to survive under high light intensities.
Salna B, Benabbas A, Sage JT, et al., 2016, Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins, Nature Chemistry, Vol: 8, Pages: 874-880, ISSN: 1755-4330
Directional proton transport along ‘wires’ that feed biochemical reactions in proteins is poorly understood. Amino-acid residues with high pKa are seldom considered as active transport elements in such wires because of their large classical barrier for proton dissociation. Here, we use the light-triggered proton wire of the green fluorescent protein to study its ground-electronic-state proton-transport kinetics, revealing a large temperature-dependent kinetic isotope effect. We show that ‘deep’ proton tunnelling between hydrogen-bonded oxygen atoms with a typical donor–acceptor distance of 2.7–2.8 Å fully accounts for the rates at all temperatures, including the unexpectedly large value (2.5 × 109 s−1) found at room temperature. The rate-limiting step in green fluorescent protein is assigned to tunnelling of the ionization-resistant serine hydroxyl proton. This suggests how high-pKa residues within a proton wire can act as a ‘tunnel diode’ to kinetically trap protons and control the direction of proton flow.
Pande K, Hutchison CDM, Groenhof G, et al., 2016, Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein, Science, Vol: 352, Pages: 725-729, ISSN: 1095-9203
Many biological processes depend on detecting and responding to light. The response is often mediated by a structural change in a protein that begins when absorption of a photon causes isomerization of a chromophore bound to the protein. Pande et al. used x-ray pulses emitted by a free electron laser source to conduct time-resolved serial femtosecond crystallography in the time range of 100 fs to 3 ms. This allowed for the real-time tracking of the trans-cis isomerization of the chromophore in photoactive yellow protein and the associated structural changes in the protein.Science, this issue p. 725A 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.
Hutchison CDM, Tenboer J, Kupitz C, et al., 2016, Photocycle populations with femtosecond excitation of crystalline photoactive yellow protein, Chemical Physics Letters, Vol: 654, Pages: 63-71, ISSN: 0009-2614
We investigate photocycle excitation of crystalline photoactive yellow protein using femtosecond laser pulses. This work establishes the feasibility and suitable optical excitation conditions to perform femtosecond time resolved X-ray crystallographic measurements using an X-ray free electron laser. Flash photolysis experiments demonstrated photocycle yields of the long-lived ‘pB’ signalling state of PYP of up to 10% with pulse durations of 130, 500 and 850 fs at 450 nm wavelength. The power density dependence of the transient pB concentration depends strongly on the pulse duration primarily because photobleaching is prominent at the GW/mm2 level.
Kim TW, Yang C, Kim Y, et al., 2016, Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein, Physical Chemistry Chemical Physics, Vol: 18, Pages: 8911-8919, ISSN: 1463-9084
Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wild-type PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix.
van Wilderen L, Silkstone G, Mason M, et al., 2015, Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen, FEBS Open Bio, Vol: 5, Pages: 885-892, ISSN: 2211-5463
Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non‐covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped‐flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033–21040 ). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH−). Reaction of this with dioxygen leads to transient radical (FADH) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.
Kaucikas M, Fitzpatrick A, Bryan E, et al., 2015, Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, Vol: 83, Pages: 397-402, ISSN: 0887-3585
Kaucikas M, Tros M, van Thor JJ, 2015, Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the dronpa fluorescent protein, The Journal of Physical Chemistry B: Biophysical Chemistry, Biomaterials, Liquids, and Soft Matter, Vol: 119, Pages: 2350-2362, ISSN: 1520-5207
The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis–trans and trans–cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright “on” and dark “off” states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the A
van Thor JJ, Madsen A, 2015, A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography, Structural Dynamics, Vol: 2, ISSN: 2329-7778
In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.
Tenboer J, Basu S, Zatsepin N, et al., 2014, Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein, Science, Vol: 346, Pages: 1242-1246, ISSN: 1095-9203
Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
Thompson LM, Champion PM, Sage JT, et al., 2014, Analytical harmonic vibrational frequencies for the green fluorescent protein computed with ONIOM: Chromophore mode character and its response to environment, 247th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Thompson LM, Lasoroski A, Champion PM, et al., 2014, Analytical Harmonic Vibrational Frequencies for the Green Fluorescent Protein Computed with ONIOM: Chromophore Mode Character and Its Response to Environment, Journal of Chemical Theory and Computation, Vol: 10, Pages: 751-766, ISSN: 1549-9618
van Thor JJ, Warren MM, Lincoln CN, et al., 2014, Signal to noise considerations for single crystal femtosecond time resolved crystallography of the Photoactive Yellow Protein, FARADAY DISCUSSIONS, Vol: 171, Pages: 439-455, ISSN: 1359-6640
Juozapavicius M, Kaucikas M, Dimitrov SD, et al., 2013, Evidence for “Slow” Electron Injection in Commercially Relevant Dye-2 Sensitized Solar Cells by Vis−NIR and IR Pump−Probe Spectroscopy, J Phys Chem C
We present femtosecond to nanosecond transient absorption (TA)data on electron injection in dye-sensitized solar cells (DSSCs) fabricated with lowvolatility, commercially relevant electrolytes, with and without added lithium.Results are shown over an extended time range (300 fs−6.3 ns) and extendedwavelength range (800−1400 nm) for both N719 and C106 dyes. Kinetics weremeasured on both TiO2 and noninjecting ZrO2. Using the latter, we havedetermined the spectra and absorption coefficient of N719* across the wavelengthrange. We find an isosbestic point in the TA spectra on TiO2 near 900 nm for allcells, existing from <1 ps to >1 ns. We show how measurements near this isosbesticpoint can give a false impression of uniformly femtosecond injection dynamics inDSSCs. Comparison of dynamics measured at 1200 nm with mid-IR transient absorption measured at 5100 nm confirms amajority proportion of slow (>10 ps) electron injection in these commercially relevant cells. We also comment on a recentpublication which appears to directly contradict the results we present.
Kim TW, Lee JH, Choi J, et al., 2013, Unravelling the protein structural dynamics of photoactive yellow protein in solution using pump-probe X-ray solution scattering
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.