88 results found
Hutchison CDM, Baxter JM, Fitzpatrick A, et al., 2023, Optical control of ultrafast structural dynamics in a fluorescent protein., Nat Chem
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.
Fadini A, Hutchison CDM, Morozov D, et al., 2023, Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism, Journal of the American Chemical Society, Vol: 145, Pages: 15796-15808, ISSN: 0002-7863
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 β-barrel across the time window of our measurements.
van Thor JJ, Champion PM, 2023, Photoacid dynamics in the green fluorescent protein, Annual Review of Physical Chemistry, Vol: 74, Pages: 1-22, ISSN: 0066-426X
The photoacid dynamics of fluorescent proteins include both electronic excited- and ground-state mechanisms of proton transfer. The associated characteristic timescales of these reactions range over many orders of magnitude, and the tunneling, barrier crossing, and relevant thermodynamics have in certain cases been linked to coherent nuclear motion. We review the literature and summarize the experiments and theory that demonstrate proton tunneling in the electronic ground state of the green fluorescent protein (GFP). We also discuss the excited-state proton-transfer reaction of GFP that takes place on the picosecond timescale. Although this reaction has been investigated using several vibrational spectroscopic methods, the interpretation remains unsettled. We discuss recent advances as well as remaining questions, in particular those related to the vibrational mode couplings that involve low-frequency modulations of chromophore vibrations on the timescale of proton transfer.
Baxter JM, Hutchison CDM, Maghlaoui K, et al., 2022, Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy, JOURNAL OF PHYSICAL CHEMISTRY B, Vol: 126, Pages: 9288-9296, ISSN: 1520-6106
Chukhutsina VU, Baxter JM, Fadini A, et al., 2022, Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization, Nature Communications, Vol: 13, Pages: 1-14, ISSN: 2041-1723
Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9’-C8’ and C7’-C6’ single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.
Fadini A, Van Thor J, 2022, Photoactivation in a fluorescent protein proceeds via the hula twist : mechanism revealed through TR-SFX, Acta Crystallographica Section A Foundations and Advances, Vol: 78, Pages: e784-e784, ISSN: 2053-2733
Browning ND, Bryan W, Clarke J, et al., 2022, The Design and Operation of a New Relativistic Ultrafast Electron Diffraction and Imaging (RUEDI) National Facility in the UK, Microscopy and Microanalysis, Vol: 28, Pages: 2764-2765, ISSN: 1431-9276
Chukhutsina VU, van Thor JJ, 2022, Molecular Activation Mechanism and Structural Dynamics of Orange Carotenoid Protein, Physchem, Vol: 2, Pages: 235-252
<jats:p>Like most photosynthetic organisms, cyanobacteria are vulnerable to fluctuations in light intensity, which can damage their photosynthetic machinery. To protect against this, they use a photoprotective mechanism called non-photochemical quenching (NPQ), where excess absorbed photo-energy is dissipated as heat. In cyanobacteria, light activation of Orange Carotenoid Protein (OCP) is the critical first step in the NPQ response. OCP is also the only known photosensitive protein, which uses carotenoid for its activation. We summarize the current knowledge on the light induced reactions of OCP; the different mechanisms of activation that have been proposed; photocycle kinetics and characteristics; and the reported structural intermediates. We discuss the possible interpretations of reported experimental results, and we formulate important open questions and directions for future work, to reveal the molecular and structural basis of photosensing by OCP.</jats:p>
Rakowski R, Fisher W, Calbo J, et al., 2022, High Power Irradiance Dependence of Charge Species Dynamics in Hybrid Perovskites and Kinetic Evidence for Transient Vibrational Stark Effect in Formamidinium, NANOMATERIALS, Vol: 12
Hutchison CDM, Fadini A, van Thor JJ, 2022, Linear and Non-Linear Population Retrieval with Femtosecond Optical Pumping of Molecular Crystals for the Generalised Uniaxial and Biaxial Systems, APPLIED SCIENCES-BASEL, Vol: 12
Hutchison CDM, Parker S, Chukhutsina V, et al., 2021, Open hardware microsecond dispersive transient absorption spectrometer for linear optical response, Photochemical and Photobiological Sciences, Vol: 21, Pages: 23-35, ISSN: 1474-905X
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.
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.
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