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.     

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.     

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.     

Molecular Dynamics Simulation of the Excited-State Dynamics of Bacteriorhodopsin

Abstract— The excited-state dynamics of bacteriorhodopsin was studied by molecular dynamics simulation. For the purpose of suppressing large displacement of amino acid residues on the surface of bacteriorhodopsin, positional restraints were imposed on these residues. A new method was developed to investigate the movement of amino acid residues upon photoexcitation and their role on the ultrafast photoisomerization of the chromophore. The structural change of bacteriorhodopsin was then traced up to 200 fs, i.e. until the formation of the intermediate I. We found that when all the conjugated bonds of the chromophore were allowed to twist freely in the excited state, many bonds including the C13=C14 bond twist in large scale within 100 fs. When only the C13=C14 bond and the single bonds were allowed to twist freely, the twisting took place at most 20° within 200 fs. From these results, it is claimed that a special potential surface is provided for the C13=C14 bond twisting by the protein environment in the course of the isomerization reaction, giving rise to the specific, ultrafast photoisomerization of bacteriorhodopsin. As a trace of such a mechanism, we observed that several functionally important residues incuding Asp85, Asp212 and Tyr185 responded quickly to the photoexcitation of the chromophore.     

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.     

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.     

Exploring the reason for increased activity of SHP2 caused by D61Y mutation through molecular dynamics

Juvenile myelomonocytic leukaemia, an aggressive myeloproliferative neoplasm, is characterized by thrombocytopenia, splenomegaly, fever and excess myelomonocytic cells. Approximately 35% of patients with JMML occur D61Y mutation in PTPN11, and it increases the activity of the protein. However, the effect of the D61Y mutation on SHP2 conformations in molecular basis is poorly understood. Therefore, the molecular dynamics simulations on SHP2-D61Y and SHP2-WT were performed to explore the effect of D61Y mutation on SHP2 and explain the reason for high activity of SHP2-D61Y mutant. The study on the RMSF, per-residue RMSD, PCA, DCCM and secondary structure found that the flexibilities of regions (residues His458-Ser460 and Gln506-Ala509) in SHP2-D61Y were higher than the corresponding regions in SHP2-WT, and the conformations of these regions almost transformed from α-helix and β-strand to Turn, respectively. Thus, the catalytical sites in the PTP domain (residues Asn217-Thr524) were exposed to the substrate easily, which contributed to the enhancement of SHP2-D61Y activity. Moreover, the residue interaction network, H bond occupancy and binding free energy were calculated, revealing that conformational difference were caused by distinctions in residue-residue interactions between Asp/Tyr61-Gln506, Gln506-Gln510, Gln506-Phe251, Gln506-Gly60, Gln506-Tyr63, Asp/Tyr61-Cys459, Cys459-Ile463 and Cys459-Arg465. The study here may offer the valuable information to explore the reason for the increased activity of SHP2 after D61Y-mutation.     

Kinetics of acid-induced spectral changes in the GFPmut2 chromophore

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.     

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   

Controlling Light-Induced Proton Transfer from the GFP Chromophore

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I⁻ and deprotonated trichloroacetic acid (TCA⁻). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S₁, revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S₁, depending on the quantum-density distribution upon vibrational excitation. We also show that the S₁ potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.     

Ultrafast exciton dynamics in CdSe quantum dots studied from bleaching recovery and fluorescence transients

We have performed ultrafast absorption bleach recovery and fluorescence upconversion measurements ( approximately 100 fs time resolution) for three CdSe samples, with nanoparticle diameters of 2.7, 2.9, and 4.3 nm. The two types of experiments provide complementary information regarding the contributions of the different processes involved in the fast relaxation of electrons and holes in the CdSe quantum dots. Transient absorption and emission experiments were conducted for the 1S [1Se-1S3/2(h)] transition, 1S(e) and 1S3/2(h) representing the lowest electron (e) and hole (h) levels. The bleach recovery of the 1S transition shows a approximately 400-500 fs initial rise, which is followed by a size-dependent approximately 10-90 ps decay and finally a long-lived (approximately ns) decay. The fluorescence upconversion signal for the 1S transition shows quite different temporal behavior: a two times slower rise time (approximately 700-1000 fs) and, when the fluorescence upconversion signal has risen to about 20% of its maximum intensity, the signal displays a slight leveling off (bend), followed by a continued rise until the maximum intensity is reached. This bend is well reproducible and power and concentration independent. Simulations show that the bend in the rise is caused by a very fast decay component with a typical time of about 230-430 fs. Considering that the 1S quantum dot excitation is comprised of five exciton substates (F=+/-2, +/-1L, 0L, +/-1U, and 0U), we attribute the disparity in the rise of the bleaching and emission transients to the results from the dynamics of the different excitons involved in respectively the bleaching and fluorescence experiments. More specifically, in transient absorption, population changes of the F=+/-1U excitons are probed, in emission population effects for the F=+/-2 ("dark") and the F=+/-1L ("bright") exciton states are monitored. It is discussed that the fast (approximately 400-500 fs) rise of the bleach recovery is representative of the feeding of the F=+/-1U exciton (by filling of the 1S(e) electron level) and that the slower (approximately 700-1000 fs) feeding of the emissive +/-2, +/-1L excitons is determined by the relaxation of the hole levels within the 1S3/2 fine structure. Finally, the approximately 230-430 fs component, typical of the bend in the fluorescence transient, is attributed to the thermalization of the close-lying +/-2 ("dark") and +/-1L ("bright") excitons.     

Protein-protein interaction of a Pharaonis halorhodopsin mutant forming a complex with Pharaonis halobacterial transducer protein II detected by Fourier-transform infrared spectroscopy

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199(ppR)-Asn74(pHtrII) and Thr189(ppR)-Glu43 (pHtrII), Ser62(pHtrII). It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L(1) and L(2) intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250(pHR) and Asn74(pHtrII) were detected for the L(1) and L(2) intermediates, supporting that Tyr250(pHR) forms a hydrogen bond with Asn74(pHtrII) as similarly to Tyr199(ppR). The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156(pHR) in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.     

Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C‐Terminal Extension of a Bacterial Flavodoxin Reductase

The role of the mobile C‐terminal extension present in Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) was evaluated using steady‐state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C‐terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild‐type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD‐binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge‐transfer quenching of ¹FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet‐ and triplet‐state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.     

Chromophore effect in photodissociation at 266 nm of protonated peptides generated by matrix-assisted laser desorption ionization (MALDI)

Chromophore effect in the photodissociation of protonated peptides at 266 nm was investigated using synthetic peptides with the sequence RGGXGGGGGR where X was a phenylalanyl(F), tyrosyl(Y), cysteinyl(C), glycyl(G), seryl(S), or histidyl(H) residue. The peptides with an F or Y residue dissociated efficiently. Fragment ions due to cleavages at either end of the chromophore were especially prominent just as for the peptide with a tryptophanyl residue reported previously.1Photodissociation was observed even for the peptides without any noticeable chromophore at 266 nm. Here, dissociation at all the peptide bonds was almost equally prominent. Photodissociation of the protonated angiotensin I was investigated using the spectral correlation rules observed in the model systems. Role of the chromophores and the plausible mechanisms involved are discussed.     

氯化钠对朊病毒结构稳定性影响的分子动力学研究

朊病毒疾病是由正常构象的PrPC转化为致病构象的PrPSc引起的一类可传染的蛋白质构象病.采用分子动力学模拟的方法研究了0～500mmol/L的NaCl溶液体系对人朊病毒构象影响并深入探讨了其分子机制.研究发现NaCl可以降低朊病毒的结构稳定性,并引起其α-螺旋含量的急剧降低.进一步的研究表明高浓度NaCl溶液体系能够显著破坏朊病毒螺旋1内部的重要盐桥Asp144-Arg148和Asp147-Arg151,同时明显降低其主要氢键Arg151 N:Asp147 O,Tyr150 N:Glu146 O,Tyr149 N:Tyr145 O和Arg148 N:Asp144 O的稳定性,并诱导朊病毒的疏水核心发生明显扩张,促使朊病毒整体稳定性的下降,这些可能是NaCl促进朊病毒构象转换的重要原因.     

Y443F mutation in the substrate-binding domain of extracellular PHB depolymerase enhances its PHB adsorption and disruption abilities

Extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ_RpiT1) from Ralstonia pickettii T1 adsorbs to the PHB surface via its substrate-binding domain (SBD) and cleaves the PHB chain using its catalytic domain. Our previous study (Biomacromolecules 2010; 11: 113-119) has suggested that the hydrophobic interaction between the amino acid residues at positions 441, 443, and 445 in the SBD and the PHB surface plays a crucial role in facilitating the association phase of the enzyme adsorption process. In the present study, in order to improve PhaZ_RpiT1 for effective PHB degradation, we targeted Tyr at position 443 for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues. We designed a mutant enzyme with an amino acid substitution at this position, taking the following factors into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. The purified Y443F enzyme showed identical CD spectrum and hydrolysis activity for a water-soluble substrate with the wild type, indicating that the mutation had no influence on the structure and the ester bond cleavage activity. In contrast, the Y443F enzyme had higher PHB degradation activity than the wild type. Kinetic analysis of PHB degradation suggests that this amino acid substitution promoted not only the adsorption of the mutant enzyme to PHB, but also the disruption of the PHB surface to enhance the hydrolysis of the PHB polymer chain.     

An analysis of ATP binding with kinase catalytic subunit by molecular dynamics simulation of the CDK2 active kinase crystal lattice

Molecular dynamics simulation was used to analyze changes in the functionally significant structural elements of the crystal lattices of pT160-CDK2/cyclin and A/ATP-Mg²⁺/substrate complexes of the native (CDK2-G16) and mutant (CDK2-S16) active kinases at physiological temperatures (300 K). The structural rearrangement of ATP caused by changes in the kinase catalytic domain was studied. ATP was fixed by the ionic and H-bond interactions of several residues, including Lys33, Asp145, and side-chain amides of the G loop between β1 and β2. The binding of the kinases to complexes with cyclin and the phosphorylation of T160 in the active complex of the CDK2 kinase result in the ATP orientation more convenient for the transfer of the phosphate group to the substrate. An analysis of interatomic distances in the ATP active site region and Asp145, Asn132, Lys33 catalytic sites participating in the orientation of ATP phosphates revealed that the Asp 145 amino acid residue was situated noticeably closer to the ATP molecule in the native complex than in its mutant counterpart. The same is true of the arrangement of the Lys33 residue with respect to ATP.     

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.     

Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature

Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.