Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs

Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS-GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804-807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS-GAF proteins and their P3 D216A, Y272F and P2 D202A PAS-GAF-PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS-GAF proteins was significantly longer than in their PAS-GAF-PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS-GAF-PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4-1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers.     

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

Photoisomerization of a protein‐bound chromophore is the basis of light sensing and signaling in many photoreceptors. Phytochrome photoreceptors can be photoconverted reversibly between the Pr and Pfr states through photoisomerization of the methine bridge between rings C and D. Ground‐state heterogeneity of the chromophore has been reported for both Pr and Pfr. Here, we report ultrafast visible (Vis) pump–probe and femtosecond polarization‐resolved Vis pump–infrared (IR) probe studies of the Pfr photoreaction in native and ¹³C/¹⁵N‐labeled Cph1 phytochrome with unlabeled PCB chromophore, demonstrating different S₀ substates, Pfr‐I and Pfr‐II, with distinct IR absorptions, orientations and dynamics of the carbonyl vibration of ring D. We derived time constants of 0.24 ps, 0.7 ps and 6 ps, describing the complete initial photoreaction. We identified an isomerizing pathway with 0.7 ps for Pfr‐I, and silent dynamics with 6 ps for Pfr‐II. We discuss different origins of the Pfr substates, and favor different facial orientations of ring D. The model provides a quantum yield for Pfr‐I of 38%, in line with ~35% ring D rotation in the electronic excited state. We tentatively assign the silent form Pfr‐II to a dark‐adapted state that can convert to Pfr‐I upon light absorption.     

Sub-picosecond mid-infrared spectroscopy of phytochrome Agp1 from Agrobacterium tumefaciens.

The photoinduced primary reaction of the biliverdin binding phytochrome Agp1 (Agp1-BV) from Agrobacterium tumefaciens was investigated by sub-picosecond time-resolved Vis pump-IR probe spectroscopy. Three time constants of tau(1)=0.7+/-0.05 ps, tau(2)=3.3+/-0.2 ps and tau(3)=33.3+/-1.5 ps could be isolated from the dynamics of structurally specific marker bands of the BV chromophore. These results together with those of accompanying sub-picosecond Vis pump-Vis probe spectroscopy allow the extension of the reaction scheme for the primary process by a vibrationally excited electronic ground state. The isomerization at the C15=C16 bond occurs within the lifetime of the excited electronic state. A quantum yield of 0.094 for the primary reaction is determined, suggesting that the quantum yield of formation of the P(fr) far-red-absorbing form is already established in the primary photoreaction of the P(r) (red-absorbing) form.     

Solvent and solvent isotope effects on the vibrational cooling dynamics of a DNA base derivative

Vibrational cooling by 9-methyladenine was studied in a series of solvents by femtosecond transient absorption spectroscopy. Signals at UV and near-UV probe wavelengths were assigned to hot ground state population created by ultrafast internal conversion following electronic excitation by a 267 nm pump pulse. A characteristic time for vibrational cooling was determined from bleach recovery signals at 250 nm. This time increases progressively in H2O (2.4 ps), D2O (4.2 ps), methanol (4.5 ps), and acetonitrile (13.1 ps), revealing a pronounced solvent effect on the dissipation of excess vibrational energy. The trend also indicates that the rate of cooling is enhanced in solvents with a dense network of hydrogen bonds. The faster rate of cooling seen in H2O vs D2O is noteworthy in view of the similar hydrogen bonding and macroscopic thermal properties of both liquids. We propose that the solvent isotope effect arises from differences in the rates of solute-solvent vibrational energy transfer. Given the similarities of the vibrational friction spectra of H2O and D2O at low frequencies, the solvent isotope effect may indicate that a considerable portion of the excess energy decays by exciting relatively high frequency (>/=700 cm-1) solvent modes.     

Resonance Raman Structural Evidence that the Cis-to-Trans Isomerization in Rhodopsin Occurs in Femtoseconds

Picosecond time-resolved resonance Raman spectroscopy is used to probe the structural changes of rhodopsin's retinal chromophore as the cis-to-trans isomerization reaction occurs that initiates vision. Room-temperature resonance Raman spectra of rhodopsin's photoproduct with time delays from -0.7 to 20.8 ps are measured using 2.2 ps, 480 nm pump and 1.5 ps, 600 nm probe pulses. Hydrogen-out-of-plane (HOOP) modes at 852, 871, and 919 cm(-1), fingerprint peaks at 1272, 1236, 1211, and 1166 cm(-1), and a broad red-shifted ethylenic band at 1530 cm(-1) are present at the earliest positive pump-probe time delay of 0.8 ps, indicating that the chromophore is already in a strained, all-trans configuration. Kinetic analyses of both the HOOP and ethylenic regions of the photoproduct spectra reveal that these features grow in with fast ( approximately 200 fs) and slow ( approximately 2-3 ps) components. These data provide the first structural evidence that photorhodopsin has a thermally unrelaxed, torsionally strained all-trans chromophore within approximately 1 ps, and possibly within 200 fs, of photon absorption. Following this ultrafast product formation, the all-trans chromophore cools and conformationally relaxes within a few picoseconds to form bathorhodopsin. This cooling process is revealed as an ethylenic frequency blue-shift of 6 cm(-1) (tau approximately 3.5 ps) as well as an ethylenic width narrowing (tau approximately 2 ps). The ultrafast production of photorhodopsin is likely accompanied by an impulsively driven, localized protein response. More delocalized protein modes are unable to relax on this ultrafast time scale enabling the chromophore-protein complex to store the large amounts of photon energy (30-35 kcal/mol) that are subsequently used to drive activating protein conformational changes.     

Proton transfer events in GFP

Proton transfer is one of the most important elementary processes in biology. Green fluorescent protein (GFP) serves as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. Illumination initiates proton transfer through a 'proton-wire', formed by the chromophore (the proton donor), water molecule W22, Ser205 and Glu222 (the acceptor), on a picosecond time scale. To obtain a more refined view of this process, we have used a combined approach of time resolved mid-infrared spectroscopy and visible pump-dump-probe spectroscopy to resolve with atomic resolution how and how fast protons move through this wire. Our results indicate that absorption of light by GFP induces in 3 ps (10 ps in D(2)O) a shift of the equilibrium positions of all protons in the H-bonded network, leading to a partial protonation of Glu222 and to a so-called low barrier hydrogen bond (LBHB) for the chromophore's proton, giving rise to dual emission at 475 and 508 nm. This state is followed by a repositioning of the protons on the wire in 10 ps (80 ps in D(2)O), ultimately forming the fully deprotonated chromophore and protonated Glu222.     

N-乙基丙酰胺的飞秒二维红外光谱

利用飞秒二维红外实验方法, 结合稳态红外光谱实验和计算化学手段, 对β-肽模型分子N-乙基丙酰胺(NEPA)的超快结构动力学进行了研究. 结果表明, 在水溶液中, NEPA具有类α-肽酰胺-I 带的振动特征, 并表现出对分子结构和化学环境的灵敏性. 二维红外光谱动力学结果揭示了一个1 ps 左右的光谱扩散时间, 与酰胺-水之间的氢键结构动力学时间尺度一致.     

Ultrafast Photo-ion Probing of the Ring-Opening Process in Trans-Stilbene Oxide

The ultrafast photo-induced ring opening of the oxirane derivative trans-stilbene oxide has been studied through the use of ultrafast UV/UV pump-probe spectroscopy by using photo-ion detection. Single- and multiphoton probe paths and final states were identified through comparisons between UV power studies and synchrotron-based vacuum ultraviolet (VUV) single-photon ionization studies. Three major time-dependent features of the parent ion (sub-450 fs decay, (1.5±0.2) ps, and >100 ps) were observed. These decays are discussed in conjunction with the primary ring-opening mechanism of stilbene oxide, which occurs through C−C dissociation in the oxirane ring. The appearance of fragments relating to the masses of dehydrogenated diphenylmethane (167 amu) and dehydrogenated methylbenzene (90 amu) were also investigated. The appearance of the 167 amu fragment could suggest an alternative ultrafast ring-opening pathway via the dissociation of one of the C−O bonds within the oxirane ring.     

Orientational motions of vibrational chromophores in molecules at the air/water interface with time-resolved sum frequency generation

The first time-resolved experiments in which interfacial molecules are pumped to excited electronic states and probed by vibrational sum frequency generation (SFG) are reported. This method was used to measure the out-of-plane rotation dynamics, i.e. time dependent changes in the polar angle, of a vibrational chromophore of an interfacial molecule. The chromophore is the carbonyl group, the rotation observed is that of the -C=O bond axis, with respect to the interfacial normal, and the interfacial molecule is coumarin 314 (C314) at the air/water interface. The orientational relaxation time was found to be 220+/-20 ps, which is much faster than the orientational relaxation time of the permanent dipole moment axis of C314 at the same interface, as obtained from pump-second harmonic probe experiments. Possible effects on the rotation of the -C=O bond axis due to the carbonyl group hydrogen bonding with interfacial water are discussed. From the measured equilibrium orientation of the permanent dipole moment axis and the carbonyl axis, and knowledge of their relative orientation in the molecule, the absolute orientation of C314 at the air/water interface is obtained.     

Unravelling the ultrafast photodecomposition mechanism of dibenzoyl peroxide in solution by time-resolved IR spectroscopy

The ultrafast photo-fragmentation of dibenzoyl peroxide (DBPO) is studied using femtosecond UV excitation at 266 nm and mid-infrared broadband probe pulses to elucidate the dissociation mechanism. With the help of (13)C-labeled DBPO it was possible to unambiguously assign transient IR bands in the fingerprint region to the benzoyloxy radical. Our experiments show that the fragmentation is controlled by the S(1)-lifetime of DBPO and within 0.4 +/- 0.2 ps leads to a benzoyloxy/phenyl radical pair plus CO(2)via concerted bond breakage of the O-O and the phenyl-C(carbonyl) bond. 20% of the radical pairs geminately recombine to phenyl benzoate on a timescale of 70 ps.     

A two-step ICT process for solvatochromic betaine pyridinium revealed by ultrafast spectroscopy, multivariate curve resolution, and TDDFT calculations

This work deals with the photophysics of a pyridinium betaine, 2-pyridin-1-yl-1H-benzimidazole (SBPa), based on a combination of steady-state, femtosecond photoionization (gas phase) and femtosecond transient absorption (solution) spectroscopic measurements, supported by (LR)-PCM-(TD)DFT calculations. Preliminary and new electrochemical results have revealed a strongly negative solvatochromic charge transfer (CT) absorption due to a S(0) → S(2) vertical transition and a weakly-solvatochromic emission due to S(1) → S(0) transition. Advanced TDDFT optimizations of the Franck-Condon states S(2)(FC) and S(1)(FC) led to two additional CT levels with planar geometry, S(2)(CT) and S(1)(CT), respectively, allowing prediction of a two-step photoinduced ICT process, i.e., S(0) → S(2)(FC) and S(2)(CT) → S(1)(CT), separated by a S(2)(FC) → S(2)(CT) back charge transfer relaxation. While the pyridinium ring is the acceptor group in both steps, two different donor groups, the benzene ring and the imidazole bridge, are involved in the excitation and internal conversion processes, respectively. Femtosecond transient absorption experiments supported by MCR-ALS decomposition confirmed indeed the contribution of two distinct CT states in the photophysics of SBPa: following excitation to the S(2)(CT) state, ultrafast production of the emissive S(1) state (the only channel observable in the gas phase) was observed to occur in competition with a further ICT process toward the S(1)(CT) state, with a time constant ranging from 300 fs to 20 ps depending on the solvent. While in aprotic media this ICT process was found to be purely solvent controlled (double polarity and viscosity dependency), in protic solvents, the influence of the hydrogen bond network has to be taken into account. Comparison with data obtained for a pre-twisted SBPa analogue led us to exclude the presence of any large-amplitude geometrical change during ICT. Analyzing the solvent dependency using the power law approach, we concluded that the S(1)(CT) state decays essentially through IC in the 3-40 ps time range whereas the emissive S(1) state decays within 130-260 ps via IC, ISC and fluorescence.     

Formation of the early photoproduct lumi-R of cyanobacterial phytochrome cph1 observed by ultrafast mid-infrared spectroscopy

The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.     

The LOV2 domain of phototropin: a reversible photochromic switch

Light, oxygen, or voltage (LOV) domains constitute a new class of photoreceptor proteins that are sensitive to blue light through a noncovalently bound flavin chromophore. Blue-light absorption by the LOV2 domain initiates a photochemical reaction that results in formation of a long-lived covalent adduct between a cysteine and the flavin cofactor. We have applied ultrafast spectroscopy on the photoaccumulated covalent adduct state of LOV2 and find that, upon absorption of a near-UV photon by the adduct state, the covalent bond between the flavin and the cysteine is broken and the blue-light-sensitive ground state is regained on an ultrafast time scale of 100 ps. We thus demonstrate that the LOV2 domain is a reversible photochromic switch, which can be activated by blue light and deactivated by near-UV light.     

Transient and stationary spectroscopy of cytochrome c: ultrafast internal conversion controls photoreduction

Photoreduction of cytochrome c (Cyt c) has been reinvestigated using femtosecond-to-nanosecond transient absorption and stationary spectroscopy. Femtosecond spectra of oxidized Cyt c, recorded in the probe range 270-1000 nm, demonstrate similar evolution upon 266 or 403 nm excitation: an ultrafast 0.3 ps internal conversion followed by a 4 ps vibrational cooling. Late transient spectra after 20 ps, from the cold ground-state chromophore, reveal a small but measurable signal from reduced Cyt c. The yield phi for Fe3+-->Fe2+ photoreduction is measured to be phi(403) = 0.016 and phi(266) = 0.08 for 403 and 266 nm excitation. These yields lead to a guess of the barrier E(f)(A) = 55 kJ mol(-1) for thermal ground-state electron transfer (ET). Nanosecond spectra initially show the typical absorption from reduced Cyt c and then exhibit temperature-dependent sub-microsecond decays (0.5 micros at 297 K), corresponding to a barrier E(A)(b) = 33 kJ mol(-1) for the back ET reaction and a reaction energy DeltaE = 22 kJ mol(-1). The nanosecond transients do not decay to zero on a second time scale, demonstrating the stability of some of the reduced Cyt c. The yields calculated from this stable reduced form agree with quasistationary reduction yields. Modest heating of Cyt c leads to its efficient thermal reduction as demonstrated by differential stationary absorption spectroscopy. In summary, our results point to ultrafast internal conversion of oxidized Cyt c upon UV or visible excitation, followed by Fe-porphyrin reduction due to thermal ground-state ET as the prevailing mechanism.     

Nature of biological water: a femtosecond study

The quasi-bound biological or structured water molecules in a protein play a key role in many biological processes. The dynamics of the biological water has been studied by femtosecond spectroscopy and large-scale computer simulations. Solvation dynamics of biological water displays an almost bulk-water like ultrafast component (approximately 1 ps) and a surprising slow component at the 100-1000 ps time scale. In this article, we discuss several examples of the ultraslow component, its possible origin and implications in biology. We show that the ultrafast (approximately 1 ps) component arises from an extended hydrogen bond network while the ultraslow component originates from binding of a water molecule to a biological macromolecule.     

The photodecomposition mechanism of tert-butyl-9-methylfluorene-9-percarboxylate: new insight from femtosecond IR spectroscopy

The ultrafast photodissociation of tert-butyl-9-methylfluorene-9-percarboxylate (TBFC) is studied by mid-infrared transient absorption spectroscopy after UV excitation at 266 nm. By means of 13C-labeled TBFC and additional DFT calculations transient IR bands in the fingerprint region were unambiguously assigned to the methylfluorenyl radical. The experiments show that the fragmentation is controlled by the S1-lifetime of TBFC and, dependent on the solvent, within 0.8-2.1 ps leads to tert-butyloxy and methylfluorenyl radicals plus CO2 via concerted bond breakage of the O-O and the fluorenyl-C(carbonyl) bond. In accordance, the CO2 quantum yield is determined to be unity.     

Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.     

Multidimensional infrared spectroscopy of water. I. Vibrational dynamics in two-dimensional IR line shapes

In this and the following paper, we describe the ultrafast structural fluctuations and rearrangements of the hydrogen bonding network of water using two-dimensional (2D) infrared spectroscopy. 2D IR spectra covering all the relevant time scales of molecular dynamics of the hydrogen bonding network of water were studied for the OH stretching absorption of HOD in D2O. Time-dependent evolution of the 2D IR line shape serves as a spectroscopic observable that tracks how different hydrogen bonding environments interconvert while changes in spectral intensity result from vibrational relaxation and molecular reorientation of the OH dipole. For waiting times up to the vibrational lifetime of 700 fs, changes in the 2D line shape reflect the spectral evolution of OH oscillators induced by hydrogen bond dynamics. These dynamics, characterized through a set of 2D line shape analysis metrics, show a rapid 60 fs decay, an underdamped oscillation on a 130 fs time scale induced by hydrogen bond stretching, and a long time decay constant of 1.4 ps. 2D surfaces for waiting times larger than 700 fs are dominated by the effects of vibrational relaxation and the thermalization of this excess energy by the solvent bath. Our modeling based on fluctuations with Gaussian statistics is able to reproduce the changes in dispersed pump-probe and 2D IR spectra induced by these relaxation processes, but misses the asymmetry resulting from frequency-dependent spectral diffusion. The dynamical origin of this asymmetry is discussed in the companion paper.     

On the unusual fluorescence properties of xanthone in water

Photo-excited xanthone is known to undergo ultrafast intersystem crossing (ISC) in the 1 ps time domain. Correspondingly, its fluorescence quantum yield in most solvents is very small ( approximately 10(-4)). Surprisingly, the quantum yield in water is 100 times larger, while ISC is still rapid ( approximately 1 ps), as seen by ultrafast pump probe absorption spectroscopy. Temperature dependent steady state and time resolved fluorescence experiments point to a delayed fluorescence mechanism, where the triplet (3)npi* state primarily accessed by ISC is nearly isoenergetic with the photo-excited (1)pipi* state. The delayed fluorescence of xanthone in water decays with a time constant of 700 ps, apparently by internal conversion between the (3)npi* state and the lowest lying triplet state (3)pipi*.     

N−H‐Type Excited‐State Proton Transfer in Compounds Possessing a Seven‐Membered‐Ring Intramolecular Hydrogen Bond

A series of compounds containing 5‐(2‐aminobenzylidene)‐2,3‐dimethyl‐3,5‐dihydro‐4H‐imidazol‐4‐one ( o ‐ABDI ) as the core chromophore with a seven‐membered‐ring N?H‐type intramolecular hydrogen bond have been synthesized and characterized. The acidity of the N?H proton and thus the hydrogen‐bond strength can be fine‐tuned by replacing one of the amino hydrogen atoms by a substituent R, the acidity increasing with increasing electron‐withdrawing strength of R, that is, in the order H<COCH₃<COPh<Tosyl<COCF₃. The tosyl and trifluoroacetyl derivatives undergo ultrafast, irreversible excited‐state intramolecular proton transfer (ESIPT) that results in proton‐transfer emission solely in the red region. Reversible ESIPT, and hence dual emission, involving the normal and proton‐transfer tautomers was resolved for the acetyl‐ and benzyl‐substituted counterparts. For o ‐ABDI , which has the weakest acidity, ESIPT is prohibited due to its highly endergonic reaction. The results clearly demonstrate the harnessing of ESIPT by modifying the proton acidity and hydrogen‐bonding strength in a seven‐membered‐ring intramolecular hydrogen‐bonding system. For all the compounds studied, the emission quantum yields are weak (ca. 10^?3) in dichloromethane, but strong in the solid form, ranging from 3.2 to 47.4 %.