Changes In pKa Research Articles

Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction.

We previously showed that treatment with tadalafil, a long-acting phosphodiesterase-5a (PDE5a) inhibitor, effectively prevented adverse left ventricular (LV) remodeling of the infarcted heart. We hypothesized that short-hairpin RNA (shRNA) therapy targeting PDE5a would simulate the effects of pharmacological intervention for treatment of postinfarction LV remodeling and dysfunction. Experimental model of myocardial infarction was developed in female mice by permanent ligation of left coronary artery. Immediately after that, an adenoviral vector encoding for shRNA sequence targeting PDE5a (Ad-shPDE5a) was injected intramyocardially, which specifically inhibited PDE5a in the heart. Four weeks later, Ad-shPDE5a treated mice showed significant mitigation of the left ventricle (LV) dilatation and dysfunction as indicated by smaller LV cavity and more preserved ejection fraction and fractional shortening. Infarction size and fibrosis were significantly reduced in Ad-shPDE5a-treated mice. Additionally, more salvaged cardiomyocytes, significantly reduced collagen contents, and higher blood vessel density were observed in Ad-shPDE5a-treated mice. The cytoprotective effects of Ad-shPDE5a were demonstrated in vitro in Ad-shPDE5a transfected cardiomyocytes cultured under oxygen glucose deprivation. Among downstream mediators of PDE5a signaling, cyclic GMP (cGMP) and cGMP-dependent protein kinase G (PKG) were activated with concomitant reduction in caspase-3 activity. However, no significant change in PKA and cAMP activities were observed in Ad-shPDE5a-treated hearts. Inhibition with shRNA improved cardiac remodeling and dysfunction by reducing infarction size and cardiac fibrosis and increased cGMP and PKG activity. These findings suggest that PDE5 inhibition with Ad-shPDE5a is a novel approach for treatment of myocardial infarction.

Development of hypoxia-sensitive Gd3+-based MRI contrast agents

Hypoxia occurs in various diseases, including cancer, ischemia, and acute and chronic vascular diseases. Here we describe the design and synthesis of the first hypoxia-sensitive MRI contrast agents, SAGds. SAGds showed a pH-dependent r1 relaxivity change associated with intramolecular chelation of the nitrogen atom of the sulfonamide moiety to the Gd3+ center. There was a correlation between the pKa of the r1 relaxivity change and the sum of the Hammett σ constants of substituents on the aromatic ring. Among the synthesized compounds, 4NO22MeOSAGd was selectively reduced to the amine by rat liver microsomes under hypoxic conditions, resulting in a 1.8-fold increment of the r1 relaxivity owing to the change in pKa of the arylsulfonamide moiety. This enhancement of the r1 relaxivity could be clearly detected in T1-weighted MR images. Thus, 4NO22MeOSAGd is a ‘smart’ MRI contrast agent for the detection of hypoxia under physiological conditions.

Prediction of pKa Value of Zinc-Bound Water in Carbonic Anhydrase

The deprotonation of zinc-bound water in carbonic anhydrase II is the rate-limiting step in the catalytic reaction of CO2-bicarbonate conversion. In order to understand the factors determining the pKa of the zinc bound water at the active site, quantum chemistry calculation together with continuum model is carried out. The pka changes due to the active site mutation are well reproduced. Additionally, the structural analysis and charge/dipole examination provide evidence that the fluctuation of the active site structure and the redistribution of the electrostatics lead to the pKa shift. Also, the distinct pka value of the same type of mutation at different site results from asymmetric ligation and different electronic environment around the zinc ion. This study not only provides insight into the molecular mechanism of the enzyme but also helps design artificial material for CO2 sequestration.

Fluorescence photoswitching based on a photochromic pKa change in an aqueous solution

Reversible fluorescence photoswitching of RSA-AZO dyad 1 was clearly demonstrated in an acidic aqueous solution. The fluorescence photoswitching mechanism is based on the reversible ring opening/closing reactions of the RSA unit induced by a photochromic pK(a) change along with the photoisomerization of the AZO unit.

Epidemiology and mutational analysis of global strains of crimean-congo haemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality. Cases are reported in several countries in Africa, Europe, the Middle East, and Asia. Phylogenetic analyses based on the virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation. In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples. We then analyzed the alignment using entries from AAIndex, the Amino Acid Index database, to identify amino acid mutations that performed significant changes in charge, pka, hydropathy and side chain volume. Finally, we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage. Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.

Acidic Environment Leads to ROS-Induced MAPK Signaling in Cancer Cells

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pHe, pHi) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pHi in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pHe. Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H2O2 enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment.

Multi-pH QSAR: II. Regression Analysis Sensitive Enough to Determine the Transition-State pKa of Human Buccal Absorption.

An analysis of published human buccal absorption data of an acid and a base at six pH values extends multi-pH QSAR to an extreme: QSAR on a single compound. The compounds analyzed are 4-n-hexylphenylacetic acid and propranolol. This analysis might be considered as much an analysis of the model as the compound, itself. Ionizable compounds undergo a pKa change in the process of absorption. Multi-pH QSAR allows determination of the pKa of the presumed rate-limiting step for absorption, which we label the transition-state pKa (pKa (TS) ). Comparison of the pKa (TS) with the corresponding aqueous and membrane pKa values provides the extent of molecular penetration at the transition state. It is 49 % of the path length for the arylacetic acid and 73 % for propranolol.

Mechanical Tuning of a Catalytic Cystein PKA in Thioredoxin

Protein function is often modulated by the protonation state of functional groups. Thioredoxin is a universally conserved disulfide reductase with wide substrate specificity, and its catalytic activity is dependent on the deprotonation of Cys32. This cysteine exhibits a pKa that is markedly lower than free cysteine, ensuring catalytic activity at physiological pH. Various electrostatic effects within the active site have been suggested to cause this pKa depression. However, a consensus on the actual pKa value is still lacking, as reported values have differed depending on the method used. We show here that the pKa of Cys32 in human thioredoxin can be modulated by its substrate. We used an atomic force microscope to detect single disulfide cleavage events in immunoglobulin substrate proteins. Integrating the data from many such events for a range of pH conditions enabled precise measurement of the rate of nucleophilic attack by thioredoxin, which is linearly dependent on nucleophile concentration. Fits to the Henderson-Hasselbalch equation allowed for sensitive titration of the Cys32 pKa. Interestingly, when altering the mechanical strain on the substrate we observed a concomitant change in the enzyme pKa. We could in this manner continuously tune the Cys32 pKa between 6.5 and 7.5. These results suggest that the conformation of the substrate influences the enzyme pKa. Electrostatic interactions in the enzyme-substrate binding interface could be responsible for this modulation. The substrate dependence of the Cys32 pKa could explain why previous reports have failed to reach a consensus on its value. Our findings may indicate a general phenomenon, which brings into question the relevance of measuring enzyme pKa's in the absence of physiological substrates.

SiRNA‐mediated GABAB receptor at early fetal rat brain upon acute and chronic ethanol exposure: Down regulation of PKA and p‐CREB expression

To observe the modulatory role of GABA(B1)R upon ethanol's effect during early brain development, we studied the effects of chronic maternal (10% ethanol during pregnancy) and acute (in vitro) ethanol exposure on the neuronal protein kinase A (PKA-α) and phosphorylation of cAMP-response element binding protein (p-CREB), using a system where GABA(B1)R were specifically knocked down in the primary cells cultured at gestational day (GD) 12.5. The results showed that upon acute and chronic ethanol treatment the GABA(B1)R expression was decreased and further decreased when GABA(B1)R was transfection with siRNA, while increased upon exposure of baclofen, and baclofen plus phaclofen treatment. PKA expression was also decreased with acute and chronic ethanol treatment, whereas it showed increase upon exposure of baclofen and baclofen with phaclofen. Furthermore, intracellular Ca(2+) concentration was increased upon ethanol, baclofen, phaclofen exposure but showed decrease in GABA(B1)R siRNA group. Finally, these effects could lead to changes of p-CREB expression, which showed same expression pattern as PKA. We speculate that GABA(B)R activity upon ethanol exposure could modulate intracellular calcium homeostasis and the expressional changes of PKA and p-CREB, which cause various negative effects on fetal brain development and modulation of GABA(B)R upon ethanol exposure may underlying cause of ethanol's effects.

Deceleration of the E1P-E2P Transition and Ion Transport by Mutation of Potentially Salt Bridge-forming Residues Lys-791 and Glu-820 in Gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P(IIC)-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK(a) modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K(+)-induced stationary currents. In Rb(+) uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E(2)-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E(1)P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E(1)P ↔ E(2)P conformational transition, thus explaining the E(1)P shift and the reduced Rb(+) transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb(+) transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E(2)P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H(+) pumping P-type ATPases.

Calculating pKa values in the cAMP‐dependent protein kinase: The effect of conformational change and ligand binding

The conformational change observed upon ligand binding and phosphorylation for the cAMP-dependent protein kinase (protein kinase A-PKA) is of high importance for the regulation of its activity. We calculate pKa values and net charges for 18 3D structures of PKA in various conformations and liganded states to examine the role of electrostatics in ligand binding and activation. We find that the conformational change of PKA takes place without any significant net proton uptake/release at all pH values, thus indicating that PKA has evolved to reduce any pH-dependent barriers to the conformational motion. We furthermore find that the binding of ligands induces large changes in the net charge of PKA at most pH values, but significantly, we find that the net charge difference at physiological pH is close to zero, thus indicating that the active-site pKa values have been preorganized for substrate binding. We are unable to unequivocally resolve the identity of the groups responsible for determining the pH-activity profile of PKA but speculate that the titration of Lys 168 or the titration of ATP itself could be responsible for the loss of activity at high pH values. Finally, we examine the effect of point mutations on the pKa values of the PKA catalytic residues and find these to be relatively insensitive to both noncharge-altering and charge-altering mutations.

Cleavages in stable tetrahedral intermediates' bonds and the role of water

AbstractUsing two‐dimensional energy diagrams, results on the formation and cleavage of detectable tetrahedral intermediates are used to predict the position of transition states involved in the cleavages of unstable and stable tetrahedral intermediates (STI). A late transition state (anti‐Hammond) for the former and synchronous cleavage for the latter are predicted and supported with experimental results. For instance, in the case of STI, intrinsic leaving abilities in the order CO > CNHCO ≫ CNH were experimentally observed, confirming the departure of these groups without full heteroatom protonation. The STI designed herein mimics serine proteases enzymes due to the thermoneutral relationship between the enzyme substrate (ES) and enzyme‐acyl tetrahedral intermediate (EATI) analogs. In the STI models, a dramatic change in pKa is observed when changing the lactam ring size due to the strain in the 3‐fused‐ring system of the STI‐H2O complex formed and exo‐anomeric effect. A plot of ΔSo (STI formation) versus pKa provides evidence for the STI–water interaction. Since only one water molecule is required to produce the effect it is also detected when using ‘pure’ CDCl3 as the solvent and therefore may also play a role in the active sites of serine proteases. Copyright © 2010 John Wiley & Sons, Ltd.

Computational analysis of missense mutations causing Snyder-Robinson syndrome.

The Snyder-Robinson syndrome is caused by missense mutations in the spermine sythase gene that encodes a protein (SMS) of 529 amino acids. Here we investigate, in silico, the molecular effect of three missense mutations, c.267G>A (p.G56S), c.496T>G (p.V132G), and c.550T>C (p.I150T) in SMS that were clinically identified to cause the disease. Single-point energy calculations, molecular dynamics simulations, and pKa calculations revealed the effects of these mutations on SMS's stability, flexibility, and interactions. It was predicted that the catalytic residue, Asp276, should be protonated prior binding the substrates. The pKa calculations indicated the p.I150T mutation causes pKa changes with respect to the wild-type SMS, which involve titratable residues interacting with the S-methyl-5'-thioadenosine (MTA) substrate. The p.I150T missense mutation was also found to decrease the stability of the C-terminal domain and to induce structural changes in the vicinity of the MTA binding site. The other two missense mutations, p.G56S and p.V132G, are away from active site and do not perturb its wild-type properties, but affect the stability of both the monomers and the dimer. Specifically, the p.G56S mutation is predicted to greatly reduce the affinity of monomers to form a dimer, and therefore should have a dramatic effect on SMS function because dimerization is essential for SMS activity.

Medium Effects on the Prototropic Equilibria of Fluorescein Fluoro Derivatives in True and Organized Solution

The stepwise ionization (H(3)R(+) <==> H(2)R <==> HR(-) <==> R(2-)) of four fluorescein fluoro derivatives was studied by visible spectroscopy. The pK(a) values were determined in water, in 50 mass % aqueous ethanol, in oil-in-water microemulsions (benzene + CTAB + pentanol-1 in water with 1.0 M KCl; CTAB = cetyltrimethylammonium bromide), and in reversed ones (water + AOT in n-octane; AOT = bis-2-ethylhexylsulphosuccinate or Aerosol OT). The medium effects, DeltapK(a), i.e., changes in pK(a) of these dyes on going from water to some other solvent systems, were rationalized by considering the tautomerism, the values of microscopic ionization constants, and the charge types of the acid-base couples. An expressed shift of the tautomeric equilibria of H(2)R toward colorless lactone was registered on going from water to both aqueous ethanol and organized solutions. While the monoanions HR(-) of 3',4',5',6'-tetrafluoro- and 2,7,3',4',5',6'-hexafluorofluorescein exist in all the systems studied as a tautomer with ionized carboxylic and nonionized hydroxy groups, in the case of 2,4,5,7-tetrafluorofluorescein, the prevalence of another tautomer was observed (COOH and O(-) groups). For 2,7-difluorofluorescein (Oregon Green 488), the partial shift of the tautomeric equilibrium of HR(-) was registered from (COO(-) and OH) in water to (COOH and O(-)) in other solvent systems. The data for the dyes located in an AOT-based pseudophase indicate that the interior of the latter exerts essential differentiation of the acid strength of the dyes, probably caused by the peculiarity of dye species location in water pools. While the state of tautomeric equilibria resembles that in nonaqueous media, the absorption maxima of R(2-) species are close to those in water. Such nonuniform influence displayed by AOT-based water droplets should be taken into account when examining them by using different molecular probes.

Folding Study of Venus Reveals a Strong Ion Dependence of Its Yellow Fluorescence under Mildly Acidic Conditions

Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of (15)N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the beta-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.

Electric Field Driven Torque in ATP Synthase

Dysfunction of mitochondrial enzymes, including ATP synthase, has been implicated in type-2 diabetes, cancer, heart disease, and neurodegenerative diseases. Our electric field driven torque (EFT) model of ATP synthase predicts a scaling law relating torque to the number of proton binding sites in the rotor (c-ring) and the proton motive force (pmf) across the mitochondrial inner membrane. When the FO complex of ATP synthase is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is also important to examine the charge distribution on the protonated c-ring and in the a-subunit, which contains the proton half-channels and acts as a stator. A self-consistent field approach is used in our calculations, based on a refinement of reported ac12 structural data. The calculations reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Implications for the EFT model will be discussed. Support was provided by R21CA133153 from NHLBI and NCI at NIH and from NSF, and by grant E-1221 from the R. A. Welch Foundation. Additional support was provided by the State of Texas through the Texas Center for Superconductivity and the Norman Hackerman Advanced Research Program.

Protein–protein interaction as a powering source of oxidoreductive reactivity

Thiol-disulfide exchange reactions between thiol-disulfide oxidoreductases (e.g. thioredoxin or Trx) and client proteins can obtain a rate several orders faster than those between chemical reagents (e.g. dithiothreitol) and client proteins. The active sites of these oxidoreductases are characterized by a CXXC motif. The XX dipeptide of Trx is GP. By altering the C-terminal X to A, K and D, it is shown that the P --> K mutation confers the largest effect on the redox potential, which it elevated by 28 mV, while the P --> D mutation displays the smallest variation. The change in pK(a) of the nucleophilic thiol also follows this trend. However, GK and GA react faster with thioredoxin reductase, exhibiting a rate rank of GK > GA > GP > GD, while the rates toward insulin and PDI follow the order GP > GA > GK > GD. The rate change spans two to three orders of magnitude. This work demonstrates that redox reactivity does not correlate simply with pK(a) and redox potential, but instead supports the important role of interaction between proteins in determining the fast reactivity and rate order of Trx. A reaction mechanism involving the transient formation of a Trx-protein binding complex is proposed for the oxidoreduction of protein thiols-disulfides. Furthermore, studies on insulin reduction show that Trx acts as an enzyme rather than a redox couple. These results provide explanations for the observed variations of the CXXC motif in PDI-like proteins as well as the conservation of the CXXC motif in Trx.

Solution Structures and Ultraviolet Raman Spectra of Purines Reveal Systematic Shifts with Change in Protonation State

Purines and pyrimidines that form the nucleobases in normal and modified DNA and RNA are known to exist in several tautomeric and ionization states. The relative populations of tautomers in solution and their precise structure are highly sensitive to ring substitution and pH. Knowing the structures in different environments is essential to understand the detailed chemistry of purine modifying enzymes such as ribosyl transferases and DNA glycosylases. We used ultraviolet Raman spectroscopy in resonance with nucleobases and exhaustive DFT calculations to reliably identify solution structures of several nucleic acid enzyme substrates at different pH and established their vibrational signatures. We find that the DNA damage lesion 8oxoguanosine is in the diketone form at physiological pH. At high pH, the anion is formed via deprotonation at N1 while the enolic form was not detected. Hypoxanthine exists as a mixture of the N7H and N9H forms though xanthine exists as a single diketone tautomer in solution. Xanthine is neutral at pH 7 but pKa changes accompany formation of the corresponding mononucleotide which is found to exist predominantly in the deprotonated form at this pH. The vibrational spectra show systematic shifts correlated with protonation state with an overall reduction in the wavenumbers of the purine ring vibrations upon deprotonation. The spectral signatures of purines established here can be used to selectively observe nucleic acid-protein interaction.

Comparison of the Binding of the Switch Regions of Cardiac Troponin-I and Skeletal Troponin-I to the Functional N-Domain of Human Cardiac Troponin-C

In the heart, there are two isoforms of troponin-I (TnI) that are developmentally regulated. The slow skeletal TnI (ssTnI) is the sole isoform expressed in the embryonic or neonatal heart, while cardiac TnI (cTnI) is expressed exclusively in the adult heart. One important distinction between ssTnI and cTnI relates to differences in myofilament Ca2+-sensitivity of force development under acidic pH conditions. Hearts expressing ssTnI show heightened Ca2+-sensitivity compared with hearts expressing cTnI under basal conditions. This isoform-specific difference is greatly accentuated during acidosis, which is often associated with ischemia of myocardial infarction. In vitro and in vivo functional studies have shown that replacement of cTnI with ssTnI results in a marked enhancement of myofilament Ca2+-sensitivity at acidic pH conditions. Recent reports have indicated that this effect can be ascribed to amino acid sequence differences in ssTnI and cTnI and in particular to a critical A162H substitution in the switch region. In this study, we have used NMR spectroscopy to examine the binding of the switch regions of ssTnI (sTnI115-131) and cTnI (cTnI147-163) to the N-domain of cardiac troponin-C (cNTnC) at physiological and acidic pH conditions. The results show that the affinity of sTnI115-131 for cNTnC•Ca2+ (KD ∼50uM) is ∼3-fold stronger than that of cTnI147-163 (KD ∼150uM), but neither are affected by a pH change from 7 to 6. The pKa of H130 in sTnI115-131 is 6.2 when free and 6.7 when bound to cNTnC•Ca2+. We have also used {1H, 15N}-HSQC NMR spectroscopy to monitor the pKa changes of cNTnC•Ca2+ from peptide free to peptide bound states. The implications of these results will be discussed in the context of structure and function of myofilament protein interactions.

Ultrafast Dynamics of Protein Proton Transfer on Short Hydrogen Bond Potential Energy Surfaces: S65T/H148D GFP.

Ultrafast proton transfer dynamics on a short H-bond in a protein were studied through the time-resolved fluorescence of the S65T/H148D green fluorescent protein (GFP) mutant. In response to the change in chromophore pK(a) upon excitation, the donor-proton-acceptor structure evolves on a sub-100 fs time scale, followed by picosecond time scale vibrational cooling and host structure reorganization.