Structural basis of fluorescence fluctuation dynamics of green fluorescent proteins in acidic environments

Green fluorescent proteins (GFPs) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change in the protein &beta-barrel is a determining step for the external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by approximately 18% over a pH range of 3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single exponential with a rotational time of phi = 17 +/- 1 ns, which indicates an intact beta-barrel with a hydrodynamic volume of 78 +/- 5 nm3. In contrast, the total fluorescence (525 +/- 50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields pKa approximately 5.9 in agreement with literature values using steady-state techniques. In contrast to the intact beta-barrel at high pH, the anisotropy of neutral S65T (at pH 相似文献   

Concerted Asynchronous Hula‐Twist Photoisomerization in the S65T/H148D Mutant of Green Fluorescent Protein

Fluorescence emission of wild‐type green fluorescent protein (GFP) is lost in the S65T mutant, but partly recovered in the S65T/H148D double mutant. These experimental findings are rationalized by a combined quantum mechanics/molecular mechanics (QM/MM) study at the QM(CASPT2//CASSCF)/AMBER level. A barrierless excited‐state proton transfer, which is exclusively driven by the Asp148 residue introduced in the double mutant, is responsible for the ultrafast formation of the anionic fluorescent state, which can be deactivated through a concerted asynchronous hula‐twist photoisomerization. This causes the lower fluorescence quantum yield in S65T/H148D compared to wild‐type GFP. Hydrogen out‐of‐plane motion plays an important role in the deactivation of the S65T/H148D fluorescent state.     

Hidden electronic excited state of enhanced green fluorescent protein

Precise two-photon absorption spectra of the green fluorescent protein (GFP) and the mutants sapphire-GFP (T203I) and enhanced GFP (S65T/F64L), as well as a model compound for the chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) were measured by multiplex two-photon absorption spectroscopy. The observed TPA bands of the anionic forms of enhanced GFP and HBDI were significantly shifted to the higher energy compared with the lowest-energy bands in one-photon absorption spectra. This result indicated the existence of a hidden electronic excited state in the vicinity of the lowest excited singlet (S1) state of the anionic form of the GFP chromophore, which is the origin of the blue shift of the two-photon absorption spectra as well as two-photon fluorescence excitation spectra.     

An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (< 1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is < or = 2.4 A. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.     

Structural features responsible for GFPuv and S147P-GFP’s improved fluorescence

Green fluorescent protein (GFP) is used as a biological marker. It is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 °C. The GFPuv (F99S/M153T/V163A) and S147P-GFP mutants mature more efficiently at room temperature than wild-type GFP, and therefore result in increased fluorescence at room temperature. Computational methods have been used to examine whether the low-energy precyclized forms of these improved GFP-mutants are preorganized so that they can more efficiently form the chromophore than the wild-type and S65T-GFP. All mutations examined (S147P, F99S, M153T, V163A and F99S/M153T/V163A) more efficiently preorganize the immature precyclized forms of GFP for chromophore formation than immature wild-type GFP. It has been proposed that Arg96 is involved in chromophore formation. Our calculations suggest that the M153T and V163A mutations in GFPuv maybe partially responsible for the increased maturation efficiency observed in GFPuv because they improve the Arg96–Tyr66 interaction. The same is true for the S147P mutation in S147P-GFP.     

Theoretical Computer‐Aided Mutagenic Study on the Triple Green Fluorescent Protein Mutant S65T/H148D/Y145F

Green fluorescent protein (GFP) mutant S65T/H148D has been proposed to host a photocycle that involves an excited‐state proton transfer between the chromophore (Cro) and the Asp148 residue and takes place in less than 50 fs without a measurable kinetic isotope effect. It has been suggested that the interaction between the unsuspected Tyr145 residue and the chromophore is needed for the ultrafast sub‐50 fs rise in fluorescence. To verify this, we have performed a computer‐aided mutagenic study to introduce the additional mutation Y145F, which eliminates this interaction. By means of QM/MM molecular dynamics simulations and time‐dependent density functional theory studies, we have assessed the importance of the Cro–Tyr145 interaction and the solvation of Asp148 and shown that in the triple mutant S65T/H148D/Y145F a significant loss in the ultrafast rise of the Stokes‐shifted fluorescence should be expected.     

Pt(II) mono-carbonyl complexes of a cyclometallating 2-(2'-thienyl)-(pyridinato-C,3N') ligand: nature and dynamics of the lowest excited state of the chloro- and thiolato-complexes

The synthesis of a cyclometallated Pt(II) thiolate carbonyl complex Pt(thpy)(CO)(mts), (thpy = 2-(2'-thienyl)-pyridinate, mts = methylthiosalicylate) is reported. A combination of emission and time-resolved infrared (TRIR) techniques revealed for both Pt(thpy)(CO)(mts) and its chloride analogue Pt(thpy)(CO)Cl the predominant intra 2-(2'-thienyl)-pyridinate 3pi pi* character of the lowest electronic excited state. The unusually short lifetime (780 ps) of the intraligand 3pi pi* lowest excited state of Pt(thpy)(CO)(mts) indicates that this electronic state is influenced by another close-lying excited state, probably charge-transfer in origin.     

Theoretical study on the photolysis mechanism of 2,3-diazabicyclo[2.2.2]oct-2-ene

A CASPT2/CASSCF study has been carried out to investigate the mechanism of the photolysis of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) under direct and triplet-sensitized irradiation. By exploring the detailed potential energy surfaces including intermediates, transition states, conical intersections, and singlet/triplet crossing points, for the first excited singlet (S(1)) and the low-lying triplet states (T(1), T(2), and T(3)), we provide satisfactory explanations of many experimental findings associated with the photophysical and photochemical processes of DBO. A key finding of this work is the existence of a significantly twisted S(1) minimum, which can satisfactorily explain the envelope of the broad emission band of DBO. It is demonstrated that the S(1) (n-pi*) intermediate can decay to the T(1) (n-pi*) state by undergoing intersystem crossing (rather inefficient) to the T(2) (pi-pi*) state followed by internal conversion to the T(1) state. The high fluorescence yield and the extraordinarily long lifetime of the singlet excited DBO are due to the presence of relatively high barriers, both for intersystem crossing and for C-N cleavage. The short lifetime of the triplet DBO is caused by fast radiationless decay to the ground state.     

Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study

The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein dynamics in general is discussed     

Excited state hydrogen transfer dynamics in substituted phenols and their complexes with ammonia: ππ*-πσ* energy gap propensity and ortho-substitution effect

Lifetimes of the first electronic excited state (S(1)) of fluorine and methyl (o-, m-, and p-) substituted phenols and their complexes with one ammonia molecule have been measured for the 0(0) transition and for the intermolecular stretching σ(1) levels in complexes using picosecond pump-probe spectroscopy. Excitation energies to the S(1) (ππ*) and S(2) (πσ*) states are obtained by quantum chemical calculations at the MP2 and CC2 level using the aug-cc-pVDZ basis set for the ground-state and the S(1) optimized geometries. The observed lifetimes and the energy gaps between the ππ* and πσ* states show a good correlation, the lifetime being shorter for a smaller energy gap. This propensity suggests that the major dynamics in the excited state concerns an excited state hydrogen detachment or transfer (ESHD/T) promoted directly by a S(1)/S(2) conical intersection, rather than via internal conversion to the ground-state. A specific shortening of lifetime is found in the o-fluorophenol-ammonia complex and explained in terms of the vibronic coupling between the ππ* and πσ* states occurring through the out-of-plane distortion of the C-F bond.     

Picosecond time-resolved infrared spectroscopic investigation of excited state dynamics in a PtII diimine chromophore

This is the first report describing the use of picosecond time-resolved infrared (TRIR) spectroscopy to probe a d8 metal chromophore, Pt(4,4'-(CO2Et)(2)-2,2'-bpy)Cl2: monitoring changes in the v(CO) vibrations allows for an assignment of the lowest excited state to an MLCT with an 8.7 ps lifetime.     

Light-driven decarboxylation of wild-type green fluorescent protein

The response of wild-type GFP to UV and visible light was investigated using steady state absorption, fluorescence, and Raman spectroscopies. As reported previously [van Thor, Nat. Struct. Biol. 2002, 9, 37-41], irradiation of GFP results in decarboxylation of E222. Here it is reported that the rate of the light-driven decarboxylation reaction strongly depends on the excitation wavelength, decreasing in the order 254 nm > 280 nm > 476 nm. The relative efficiencies of decarboxylation are explained in terms of the Kolbe-type mechanism in which the excited state of the chromophore acts as an oxidant by accepting an electron from E222. Specifically, it is proposed that 254 nm excitation populates the S2 (or higher) excited state of the chromophore, whereas 404 and 476 nm excitation populate the S1 excited state of neutral and anionic forms, respectively, and that the relative oxidizing power of the three excited states controls the rate of the decarboxylation reaction. In addition, the role of W57 in the photophysics of GFP has been probed by mutating this residue to phenylalanine. These studies reveal that while W57 does not affect decarboxylation, this residue is involved in resonance energy transfer with the chromophore, thereby partially explaining the green fluorescence observed upon UV irradiation of wild-type GFP. Finally, comparison of Raman spectra obtained from nonilluminated and decarboxylated forms of wild-type GFP has provided further vibrational band assignments for neutral and anionic forms of the chromophore within the protein. In addition, these spectra provide valuable insight into the specific interactions between the protein and the chromophore that control the optical properties of wild-type GFP.     

Polarization Dependent Time-Resolved Infrared Spectroscopy and Its Applications

Polarization dependent time-resolved infrared (TRIR) spectroscopy has proven to be a useful technique to study the structural dynamics in a photochemical process. The angular information of transient species is obtainable in this measurement, which makes it a valuable technique for the investigation of electron distribution, molecular structure, and conformational dynamics. In this review, we briefly introduce the principles and applications of polarization dependent TRIR spectroscopy. We mainly focused on the following topics: (i) an overview of TRIR spectroscopy, (ii) principles of TRIR spectroscopy and its advantages compared to the other ultrafast techniques, (iii) examples that use polarization dependent TRIR spectroscopy to probe a variety of chemical and dynamical phenomena including protein conformational dynamics, excited state electron localization, and photoisomerization, (iv) the limitations and prospects of TRIR spectroscopy.     

Intra- and intermolecular electronic relaxation of the second excited singlet and the lowest excited triplet states of 1,3-dimethyl-4-thiouracil in solution

Intramolecular processes of deactivation of 1,3-dimethyl-4-thiouracil (DMTU) from the second excited singlet (S2) (pi, pi*) and the lowest excited triplet (T1) (pi, pi*) states have been studied using perfluoro-1,3-dimethylcyclohexane (PFDMCH) as a solvent. The spectral and photophysical (PP) properties of DMTU in CCl4, hexane and water have also been described. For the first time, the fluorescence from S2 state DMTU has been observed. The picosecond lifetime of DMTU in the S2 state (tau(S2)) in PFDMCH has been proposed to be determined by a very fast intramolecular reversible process of hydrogen abstraction from the ortho methyl group by the thiocarbonyl group. The shortening of tau(S2) in CCl4 is interpreted to be caused by the intermolecular interactions between DMTU (S2) and the solvent. Results of the phosphorescence decay as a function of DMTU concentration were analyzed using the Stern-Volmer formalism, which enabled determination of the intrinsic lifetime of the T1 state (tau0(T1)) and rate constants of self-quenching (k(sq)). The lifetimes, tau0(T1), of DMTU in PFDMCH and CCl4 are much longer than the values hitherto obtained in more reactive solvents. The PP properties of DMTU both in the S2 and T1 states have been shown to be determined by the thiocarbonyl group.     

PtII diimine chromophores with perfluorinated thiolate ligands: nature and dynamics of the charge-transfer-to-diimine lowest excited state

The synthesis of new Pt(II) diimine complexes bearing perfluorinated thiolate ligands, Pt(II)(NN)(4-X-C(6)F(4)-S)(2), where NN = 2,2'-bipyridine or 1,10-phenanthroline and X = F or CN, is reported, together with an investigation of the nature and dynamics of their lowest excited states. A combined UV-vis, (spectro)electrochemical, resonance Raman, and time-resolved infrared (TRIR) study has suggested that the HOMO is mainly composed of thiolate(pi)/S(p)/Pt(d) orbitals and that the LUMO is largely localized on the pi*(diimine) orbital, thus revealing the [charge-transfer-to-diimine] nature of the lowest excited state. An enhancement of the thiolate ring vibrations, C-F vibrations, and the vibration of the CN-substituent on the thiolate moiety was observed in the resonance Raman spectra, whereas no such enhancement was seen for the nonfluorinated analogues. Thus, the introduction of fluorine substituents on the thiolate moiety probably leads to a more pronounced contribution of the intrathiolate modes to the HOMO compared to the analogous complexes with nonfluorinated thiolates. Furthermore, the introduction of the p-CN group into the thiolate moiety has allowed the dynamics of the lowest excited state of Pt(bpy)(4-CN-C(6)F(4)-S)(2) to be monitored by picosecond TRIR spectroscopy. The dynamics of the lowest [charge-transfer-to-diimine] excited state are governed by ca. 2-ps vibrational cooling and 35-ps back electron transfer.     

Time-resolved optical and infrared spectral studies of intermediates generated by photolysis of trans-RhCl(CO)(PR3)2. Roles Played in the Photocatalytic Activation of Hydrocarbons

Described are picosecond and nanosecond time-resolved optical (TRO) spectral and nanosecond time-resolved infrared (TRIR) spectral studies of intermediates generated when the rhodium(I) complexes trans-RhCl(CO)L2 (L = PPh3 (I), P(p-tolyl)3 (II), or PMe3 (III)) are subjected to photoexcitation. Each of these species, which are precursors in the photocatalytic activation of hydrocarbons, undergoes CO labilization to form an intermediate concluded to be the solvated complex RhCl(Sol)L2 (A(i)). The picosecond studies demonstrate that an initial transient is formed promptly (<30 ps), which decays to A(i) with lifetimes ranging from 40 to 560 ps depending upon L and the medium. This is proposed on the basis of ab initio calculations to be a metal-to-ligand charge transfer (MLCT) excited state. Second-order rate constants (kCO) for reaction of the A(i) with CO were determined, and these depend on the nature of L and the solvent, the slowest rate being for A(I) in tetrahydrofuran (kCO = 7.1 x 10(6) M(-1) x s(-1)), the fastest being for A(III) in dichloromethane (1.3 x 10(9) M(-1) x s(-1)). Each A(i) also undergoes competitive unimolecular reaction with solvent to form long-lived transients with TRIR properties suggesting these to be Rh(III) products of oxidative addition. Although this was mostly suppressed by the presence of higher concentrations of CO (which trapped A(i) to re-form the starting complexes in each case), both TRO and TRIR experiments indicate that a fraction of the oxidative addition could not be quenched. Thus, the short-lived MLCT state or a vibrationally hot species formed during the decay of this excited state appears to participate directly in C-H activation.     

Ultrafast internal conversion of excited cytosine via the lowest pipi electronic singlet state

Computational evidence at the CASPT2 level supports that the lowest excited state pipi* contributes to the S1/S0 crossing responsible for the ultrafast decay of singlet excited cytosine. The computed radiative lifetime, 33 ns, is consistent with the experimentally derived value, 40 ns. The nOpi* state does not play a direct role in the rapid repopulation of the ground state; it is involved in a S2/S1 crossing. Alternative mechanisms through excited states pisigma* or nNpi* are not competitive in cytosine.     

Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.     

Ultrafast infrared spectral fingerprints of vitamin B12 and related cobalamins

Vitamin B(12) (cyanocobalamin, CNCbl) and its derivatives are structurally complex and functionally diverse biomolecules. The excited state and radical pair reaction dynamics that follow their photoexcitation have been previously studied in detail using UV-visible techniques. Similar time-resolved infrared (TRIR) data are limited, however. Herein we present TRIR difference spectra in the 1300-1700 cm(-1) region between 2 ps and 2 ns for adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), CNCbl, and hydroxocobalamin (OHCbl). The spectral profiles of all four cobalamins are complex, with broad similarities that suggest the vibrational excited states are related, but with a number of identifiable variations. The majority of the signals from AdoCbl and MeCbl decay with kinetics similar to those reported in the literature from UV-visible studies. However, there are regions of rapid (<10 ps) vibrational relaxation (peak shifts to higher frequencies from 1551, 1442, and 1337 cm(-1)) that are more pronounced in AdoCbl than in MeCbl. The AdoCbl data also exhibit more substantial changes in the amide I region and a number of more gradual peak shifts elsewhere (e.g., from 1549 to 1563 cm(-1)), which are not apparent in the MeCbl data. We attribute these differences to interactions between the bulky adenosyl and the corrin ring after photoexcitation and during radical pair recombination, respectively. Although spectrally similar to the initial excited state, the long-lived metal-to-ligand charge transfer state of MeCbl is clearly resolved in the kinetic analysis. The excited states of CNCbl and OHCbl relax to the ground state within 40 ps with few significant peak shifts, suggesting little or no homolysis of the bond between the Co and the upper axial ligand. Difference spectra from density functional theory calculations (where spectra from simplified cobalamins with an upper axial methyl were subtracted from those without) show qualitative agreement with the experimental data. They imply the excited state intermediates in the TRIR difference spectra resemble the dissociated states vibrationally (the cobalamin with the upper axial ligand missing) relative to the ground state with a methyl in this position. They also indicate that most of the TRIR signals arise from vibrations involving some degree of motion in the corrin ring. Such coupling of motions throughout the ring makes specific peak assignments neither trivial nor always meaningful, suggesting our data should be regarded as IR spectral fingerprints.