Photophysics of the red-form Kaede chromophore.

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.

The Effect of Conjugation on the Competition between Internal Conversion and Electron Detachment: A Comparison between Green Fluorescent and Red Kaede Protein Chromophores.

Kaede, an analogue of green fluorescent protein (GFP), is a green-to-red photoconvertible fluorescent protein used as an in vivo "optical highlighter" in bioimaging. The fluorescence quantum yield of the red Kaede protein is lower than that of GFP, suggesting that increasing the conjugation modifies the electronic relaxation pathway. Using a combination of anion photoelectron spectroscopy and electronic structure calculations, we find that the isolated red Kaede protein chromophore in the gas phase is deprotonated at the imidazole ring, unlike the GFP chromophore that is deprotonated at the phenol ring. We find evidence of an efficient electronic relaxation pathway from higher-lying electronically excited states to the S1 state of the red Kaede chromophore that is not accessible in the GFP chromophore. Rapid autodetachment from high-lying vibrational states of S1 is found to compete efficiently with internal conversion to the ground electronic state.

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii.

Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the ß5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling.

BLUF domain function does not require a metastable radical intermediate state.

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto-enol tautomerization induced by electronic excitation of the flavin ring are considered.

Proteins in action: femtosecond to millisecond structural dynamics of a photoactive flavoprotein.

Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The mechanism, the underlying structural dynamics, and the time scales for regulation of this response are central questions in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins, which is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been made over 10 decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) localized dynamics occur on the pico- to nanosecond time scale, while subsequent protein structural reorganization is observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which suppresses biological function is shown to 'short circuit' this structural relaxation pathway, suppressing the changes which occur further away from the chromophore while accelerating dynamics close to it.

Protein photochromism observed by ultrafast vibrational spectroscopy.

Photochromic proteins, such as Dronpa, are of particular importance in bioimaging and form the basis of ultraresolution fluorescence microscopy. The photochromic reaction involves switching between a weakly emissive neutral trans form of the chromophore (A) and its emissive cis anion (B). Controlling the rates of switching has the potential to significantly enhance the spatial and temporal resolution in microscopy. However, the mechanism of the switching reaction has yet to be established. Here we report a high signal-to-noise ultrafast transient infrared investigation of the photochromic reaction in the mutant Dronpa2, which exhibits facile switching behavior. In these measurements we excite both the A and B forms and observe the evolution in the IR difference spectra over hundreds of picoseconds. Electronic excitation leads to bleaching of the ground electronic state and instantaneous (subpicosecond) changes in the vibrational spectrum of the protein. The chromophore and protein modes evolve with different kinetics. The chromophore ground state recovers in a fast nonsingle-exponential relaxation, while in a competing reaction the protein undergoes a structural change. This results in formation of a metastable form of the protein in its ground electronic state (A'), which subsequently evolves on the time scale of hundreds of picoseconds. The changes in the vibrational spectrum that occur on the subnanosecond time scale do not show unambiguous evidence for either proton transfer or isomerization, suggesting that these low-yield processes occur from the metastable state on a longer time scale and are thus not the primary photoreaction. Formation of A', and further relaxation of this state to the cis anion B, are relatively rare events, thus accounting for the overall low yield of the photochemical reaction.

Ultrafast excited state dynamics of the green fluorescent protein chromophore and its kindling fluorescent protein analogue.

Fluorescent proteins exhibit a very diverse range of photochemical behaviour, from efficient fluorescence through photochromism to photochemical reactivity. Remarkably this diverse behaviour arises from chromophores which have very similar structures. Here we describe measurements and modelling of the excited state dynamics in the chromophores of GFP (HBDI) and the kindling fluorescent protein, KFP (FHBMI). The methods are ultrafast fluorescence spectroscopy with sub 50 fs time resolution and the modelling is based on the Smoluchowski equation. The excited state decays of both chromophores are very fast, longer for their anions than for the neutral form and independent of wavelength. Detailed studies show the mean fluorescence wavelength to be independent of time. The excited state decay times are also observed to be a very weak function of solvent polarity and viscosity. These results are modelled utilising recently calculated potential energy surfaces for the ground and excited states as a function of the twist coordinates about the two bridging bonds of the chromophore. For FHBMI and the scarce data on the neutral HBDI the calculations are not successful suggesting the need for refinement of these potential energy surfaces. For HBDI in methanol the simulation is successful provided a strong dependence of the radiationless decay rate on the coordinate is assumed. Such dependence should be included in future calculations of excited state dynamics. When the simulations are extended to more viscous solvents they fail to reproduce the observed weak viscosity dependence. The implications of these results for the nature of the coordinate leading to radiationless decay in the chromophore and for the photodynamics of fluorescent proteins are discussed.

Enhanced room temperature magnetoresistance and spin injection from metallic cobalt in Co/ZnO and Co/ZnAlO films.

Co/ZnO and Co/ZnAlO films were prepared by depositing ultrathin cobalt layers and semiconductor layers on glass substrates at room temperature. The films consist of metallic Co particles, semiconductor matrix, and an interfacial magnetic semiconductor with the substitution of Co(2+) for Zn(2+) in the ZnO lattice at the interface between Co particles and the semiconductor matrix. Large room temperature negative tunneling magnetoresistance was observed in the films. In addition, the magnetism and magnetoresistance were obviously enhanced by adding aluminum to the ZnO, and in one Co/ZnAlO sample, the room temperature negative magnetoresistance value reaches -12.3% at 18 kOe (compared with -8.4% of the corresponding Co/ZnO film) and the spin polarization of the tunneling electrons is about 37.5% which is characteristic of metallic Co. This enhancement of the tunneling spin polarization has been ascribed to the tunneling through an interfacial magnetic semiconductor, which causes the robust spin injection from cobalt metal into the semiconductors at room temperature resulting from the spin filter effect of the interfacial magnetic semiconductors.

Ultrafast dynamics in the power stroke of a molecular rotary motor.

Light-driven molecular motors convert light into mechanical energy through excited-state reactions. Unidirectional rotary molecular motors based on chiral overcrowded alkenes operate through consecutive photochemical and thermal steps. The thermal (helix inverting) step has been optimized successfully through variations in molecular structure, but much less is known about the photochemical step, which provides power to the motor. Ultimately, controlling the efficiency of molecular motors requires a detailed picture of the molecular dynamics on the excited-state potential energy surface. Here, we characterize the primary events that follow photon absorption by a unidirectional molecular motor using ultrafast fluorescence up-conversion measurements with sub 50 fs time resolution. We observe an extraordinarily fast initial relaxation out of the Franck-Condon region that suggests a barrierless reaction coordinate. This fast molecular motion is shown to be accompanied by the excitation of coherent excited-state structural motion. The implications of these observations for manipulating motor efficiency are discussed.

Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore.

Cis-trans photoisomerization is proposed as a key process in the photoswitching of some photoactivatable fluorescent proteins. Here we present ultrafast fluorescence measurements of the model GFP chromophore (HBDI) in the cis state and in a mixture of the cis and trans states. Our results demonstrate that the mean lifetimes of the cis and trans states are remarkably similar. Therefore, the specific isomer of the chromophore cannot be solely responsible for the different photophysics of the bright and dark states of photoactive proteins, which must therefore be due to differential interactions between the different isomers of the chromophore and the protein.