Publications
93 results found
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 [1]). 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
- Author Web Link
- Cite
- Citations: 6
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
- Author Web Link
- Cite
- Citations: 13
Kaucikas M, Warren M, Michailovas A, et al., 2013, Beam patterns in OPO employing walk-off compensating BBO crystals, Laser Physics, Pages: 1-6
Warren M, Kaucikas M, Fitzpatrick A, et al., 2013, Ground state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa, Nature Communications
Kaucikas M, Barber J, van Thor JJ, 2013, Polarization sensitive ultrafast mid-IR pump probe micro-spectrometer with diffraction limited spatial resolution, Optics Express, Vol: 21, Pages: 8357-8370
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
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.
van Thor JJ, Lincoln CN, Kellner B, et al., 2012, Ultrafast vibrational dynamics of parallel excited state proton transfer reactions in the Green Fluorescent Protein, VIBRATIONAL SPECTROSCOPY, Vol: 62, Pages: 1-6, ISSN: 0924-2031
- Author Web Link
- Cite
- Citations: 9
Lincoln CN, Fitzpatrick AE, van Thor JJ, 2012, Photoisomerisation Quantum Yield and Non-linear Cross-Sections with Femtosecond Excitation of the Photoactive Yellow Protein, Phys Chem Chem Phys, Pages: 15752-15764
Juozapavicius M, Kaucikas M, van Thor JJ, et al., 2012, Observation of Multi-Exponential Pico to Sub-Nanosecond Electron Injection in Optimized Dye-Sensitized Solar Cells With Visible-Pump Mid-Infrared-Probe Transient Absorption Spectroscopy, J. Phys. Chem. C
Kim TW, Lee JH, Choi J, et al., 2012, Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump−Probe X-ray Solution Scattering., J. Am. Chem. Soc.
Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump−probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.
Fitzpatrick AE, Lincoln CN, van Wilderen LJ, et al., 2011, Pump-Dump-Probe and Pump-Repump-Probe Ultrafast Spectroscopy Resolves Cross Section of an Early Ground State Intermediate and Stimulated Emission in the Photoreactions of the Pr Ground State of the Cyanobacterial Phytochrome Cph1., The Journal of Physical Chemistry B
The primary photoreactions of the red absorbing ground state (Pr) of the cyanobacterial phytochrome Cph1 from Synechocystis PC 6803 involve C15=C16 Z-E photoisomerisation of its phycocyanobilin chromophore. The first observable ground state intermediate in pump-probe measurements of the photocycle, 'Lumi-R', is formed with picosecond kinetics, and involves excited state decay reactions that have 3 and 14 ps time constants. Here, we have studied the photochemical formation of the Lumi-R intermediate using multi pulse picosecond visible spectroscopy. Pump-dump-probe (PDP) and pump-repump-probe (PRP) experiments were carried out by employing two femtosecond visible pulses with 1, 14 and 160 ps delays, together with a broadband dispersive visible probe. The time delays between the two excitation pulses have been selected to allow interaction with the dominant (3 and 14 ps) kinetic phases of Lumi-R formation. The frequency dependence of the PDP and PRP amplitudes was investigated at 620 nm, 640 nm, 660 nm and 680 nm, covering excited state absorption (λmax = 620 nm), ground state absorption (λmax = 660 nm) and stimulated emission (λmax = 680 nm) cross sections. Experimental double difference transient absorbance signals (∆∆OD), from the PDP and PRP measurements required corrections to remove contributions from ground state repumping. The sensitivity of the resulting ∆∆OD signals was systematically investigated for possible connectivity schemes and photochemical parameters. When applying a homogeneous (sequentially decaying) connectivity scheme in both the 3 and 14 ps kinetic phases, evidence for repumping of an intermediate that has an electronic ground state configuration (GSI) is taken from the dump-induced S1 formation with 620, 640 and 660 nm wavelengths and 1 and 14 ps repump delays. Evidence for repumping a GSI is also seen, for the same excitation wavelengths, when imposing a target connectivity scheme proposed in refs1,2 for the 1 ps repump d
Ramachandran PL, Lovett JE, Carl PJ, et al., 2011, The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 133, Pages: 9395-9404, ISSN: 0002-7863
- Author Web Link
- Cite
- Citations: 73
van Wilderen LJG, Lincoln CN, van Thor JJ, 2011, Modelling multi-pulse population dynamics from ultrafast spectroscopy, PLoS One, Vol: 6, Pages: 1-14, ISSN: 1932-6203
Current advanced laser, optics and electronics technology allows sensitive recording of molecular dynamics, from single resonance to multi-colour and multi-pulse experiments. Extracting the occurring (bio-) physical relevant pathways via global analysis of experimental data requires a systematic investigation of connectivity schemes. Here we present a Matlab-based toolbox for this purpose. The toolbox has a graphical user interface which facilitates the application of different reaction models to the data to generate the coupled differential equations. Any time-dependent dataset can be analysed to extract time-independent correlations of the observables by using gradient or direct search methods. Specific capabilities (i.e. chirp and instrument response function) for the analysis of ultrafast pump-probe spectroscopic data are included. The inclusion of an extra pulse that interacts with a transient phase can help to disentangle complex interdependent pathways. The modelling of pathways is therefore extended by new theory (which is included in the toolbox) that describes the finite bleach (orientation) effect of single and multiple intense polarised femtosecond pulses on an ensemble of randomly oriented particles in the presence of population decay. For instance, the generally assumed flat-top multimode beam profile is adapted to a more realistic Gaussian shape, exposing the need for several corrections for accurate anisotropy measurements. In addition, the (selective) excitation (photoselection) and anisotropy of populations that interact with single or multiple intense polarised laser pulses is demonstrated as function of power density and beam profile. Using example values of real world experiments it is calculated to what extent this effectively orients the ensemble of particles. Finally, the implementation includes the interaction with multiple pulses in addition to depth averaging in optically dense samples. In summary, we show that mathematical modelling is es
Sage JT, Zhang Y, McGeehan J, et al., 2011, Infrared protein crystallography, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, Vol: 1841, Pages: 760-777
We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO2. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Thor JJV, 2011, Photoconversion of the Green Fluorescent Protein and Related Proteins, Springer Series on Fluorescence, Editors: Wolfbeis, Hof
This review focuses on the mechanistic details of photochromic reactions of the green fluorescent protein (GFP) and also of its mutant derivatives and related fluorescent proteins. A number of distinct photochromic processes have so far been identified that have entirely different photochemical and chemical basis, which will be reviewed. In addition to bright fluorescence, the GFP from the jellyfish Aequorea victoria undergoes photochromic transformation with blue or UV illumination. The associated change in electronic absorption provides a spectroscopic contrast that can be used in fluorescence microscopy application to tag and track the movement of populations that are photoconverted. Key to the successful use of photoconversion for such microscopy experiments is in fact the relatively low quantum yield of the irreversible process. In the wild-type GFP, photoconversion is triggered by light-induced electron transfer from the buried anionic carboxylate of Glu222 to the optically excited protonated chromophore. An unstable carboxylate radical subsequently cleaves off a CO2 molecule in a “Kolbe” type reaction that has been trapped in a partially oriented site near the chromophore-binding site at 100K, as observed by low-temperature X-ray crystallography and cryo-infrared crystallography. Structural intermediates in the subsequent relaxation pathway involve motion of CO2, amino acids and H-bonded waters both in the chromophore vicinity and at longer range. This review provides an overview of the molecular characterisation using structural and spectroscopy methods of this photoconversion reaction of GFP. In addition, the mechanisms of photochromic reactions of mutants of GFP and related fluorescent proteins will be summarised and discussed. These include the cis–trans isomerisation and protonation changes in Dronpa, asFP595 and IrisFP and related proteins, light-induced maturation in aceGFPL, and photoinduced beta-elimination and backbone cleavage tha
Ramachandran PL, Lovett JE, Carl PJ, et al., 2011, Catching a short-lived photoreceptor intermediate with pulsed X-rays, ESRF Highlights 2011
Ramachandran P, Lovett JE, Carl P, et al., 2011, ESRF 2011 Highlight: Catching a short-lived photoreceptor intermediate with pulsed X-rays
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.