Internal Proton Research Articles

Mapping Proton Wires in Proteins: Carbonic Anhydrase and GFP Chromophore Biosynthesis

We have developed an algorithm for mapping proton wires in proteins and applied it to the X-ray structures of human carbonic anhydrase II (CA-II), the green fluorescent protein (GFP), and some of their mutants. For both proteins, we find more extensive proton wires than typically reported. In CA-II the active site wire exits to the protein surface, and leads to Glu69 and Asp72, located on an electronegative patch on the rim of the active site cavity. One possible interpretation of this observation is that positively charged, protonated buffer molecules dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction. In GFP we find a new internal proton wire, in addition to the previously reported wire involved in excited state proton transfer. The new wire is located on the other face of the chromophore, and we conjecture that it plays a role in chromophore biosynthesis that occurs following protein folding. In the last step of this process, transient carbanion formation was suggested to occur on the bridge carbon [Pouwels et al. Biochemistry 2008, 47, 10111]. Residues on the new wire (Thr62, His181, Arg96) may participate in proton abstraction from this bridge carbon atom. A possible mechanism involves a rotation of the Thr62 side chain and completion of a short wire by which the proton is transported to His181, while the negative charge is transferred to the imidazolone carbonyl, producing a homoenolate intermediate that is stabilized by Arg96. Finally, comparison of the proton wires in the two proteins reveals common motifs, such as short internalized Ser/Thr-Glu hydrogen-bonded pairs for ultrafast proton abstraction, and threonine side chain rotation functioning as a proton wire switch.

Influence of temperature and crown ether complex formation on the charge partitioning between z and c fragments formed after electron capture by small peptide dications

Electron capture by peptide dications results in N–C α bond cleavage to give c + and z or c and z + fragments. In this work we have investigated how crown ether (18-crown-6 = CE) complex formation and a change in the internal energy affect the charge division between the z and c fragments. Both complex formation and a high temperature have the effect of breaking internal ionic hydrogen bonds. The crown ether complex also lowers the probability of internal proton transfer between the two fragments, and reduces the recombination energy of the charged group it targets. The systems under study were doubly protonated di- and tripeptides, [AK+2H] 2+, [AR+2H] 2+, [KK+2H] 2+ and [GHK+2H] 2+ (A = alanine, K = lysine, R = arginine, G = glycine and H = histidine). For crown ether complexes the formation of z + ions was always preferred over c + ions. In the case of [GHK+2H] 2+, the bare ion dissociated into z 2 + + c 1 and z 1 + c 2 + from cleavage of the first and second N–C α bond, respectively, whereas z 1 + fragment ions had higher yield than c 2 + for [GHK+2H] 2+(CE). The internal energy of the ions was changed by storing them in a 22-pole ion trap in which they were equilibrated to a temperature between −60 and 90 °C in collisions with helium gas. The average internal energy increased by about 0.4 eV from the lowest to the highest temperature for the dipeptides and 0.6 eV for the tripeptide. More fragmentation occurred at the higher temperature, as observed by an increase in the formation of b + and y + ions after breakage of the peptide bond of vibrationally hot even-electron cations and from secondary reactions of z + radical cations within the time window of the experiment. However, the z + to c + partitioning was not found to depend significantly on temperature in the measured range. In addition the decay of [GHK+H] +/[GHK+2H] + and [AK+H] + formed after electron capture by [GHK+2H] 2+ and [AK+2H] 2+ was found to occur on a microsecond to millisecond timescale. The data are well described by a power-law decay, which implies that the internal energy distribution is broad after electron capture and/or hydrogen loss.

Mechanism of Internal Proton Transfer Reactions in Proteins

Proton transfer reactions are crucial in a large array of biomolecular processes, encompassing bioenergetics, biological signaling, and enzymatic catalysis. We performed a proof of concept study regarding the mechanism of internal proton transfer reactions between buried groups in proteins. A model system, that resembles the active site structure of a PAS domain bacterial photoreceptor protein, is employed in our study. A first principles approach without adjustable parameters was used to identify the energy landscape for internal proton transfer. We will report the fundamental aspects (structure, energetics, and kinetics) of the proton transfer mechanism from our study. It is expected that this mechanism may be applied to a broad range of proton transfer systems.

The photochemistry of fluorescent proteins: implications for their biological applications

Green fluorescent protein from Aequorea victoria, its relatives and derivatives are ubiquitous in their use as biological probes. In this tutorial review, we discuss the photochemistry of this fascinating class of proteins and illustrate some of their advantages and drawbacks in a range of applications. In particular, we focus on the ionisation states of the chromophore and how they are affected by internal and external proton transfer. Light-induced reversible and irreversible events are discussed in terms of the underlying chromophore structure. These phenomena have an influence on the interpretation of FRET (Förster resonance energy transfer), FRAP (fluorescence recovery after photobleaching), as well as single molecule studies.

Two protonation switches control rhodopsin activation in membranes

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to approximately 50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.

ESIPT from S2 singlet state in 3-hydroxyflavone

Spectra of the dual fluorescence of 3-hydroxyflavone in ethyl acetate with picoseconds time resolution were obtained at excitation by tunable picoseconds pulses of the optical parametric generator within S 1 and S 2 bands of absorption. Spectra dynamics reveals time development of the internal proton transfer in the excited state of molecule from the hydroxyl to the carbonyl group. The relative contribution of the tautomer form to integral emission remains essentially higher during all interval of emission observation. The obtained data directly evidence an additional channel of the internal proton transfer from the S 2 singlet state of 3-hydroxyflavone with the very high kinetic rate of 0.8 × 10 12 s −1.

Rotational symmetry of the molecular potential energy in the Cartesian coordinates

We consider the molecular Born-Oppenheimer potential energy as a function of atomic Cartesian coordinates and discuss the non-stationary Hessian properties arising due to rotational symmetry. A connection with the extended Hessian theory is included. New applications of Cartesian representation for examining and correcting raw numerical Hessian data and a simple formulation of harmonic vibrational analysis of partially optimized systems are proposed. Exemplary calculations for the porphyrin molecule with an internal proton transfer are reported. We also develop the normal transformation method to incorporate the rotational symmetry into the approximate analytical potentials, which are parametrized in the Cartesian coordinates. The transformation converts the coordinates from the space fixed frame to the frame which translates and rotates with the molecule and is determined by the Eckart conditions. New simple analytical formulas for the first and second derivatives of the transformed potential are derived. This fast method can be used to calculate the potential and its derivatives in the simulations of chemical reaction dynamics in the space fixed Cartesian frame without the need to constrain the molecular rotation or to define the local non-redundant internal coordinates.

Unusually Slow Intermolecular Proton-Deuteron Exchange in Porphycene

Abstract The rates of the intermolecular exchange of two internal protons by deuterons have been determined for alcohol solutions of porphycene and its 2,7,12,17-tetra-t-butyl derivative using femtosecond pump-probe polarization spectroscopy. The process is very slow, requiring approximately twenty hours to substitute both protons by deuterons in more than 90% of the molecules dissolved in bulk EtOD or BuOD at 293 K. Equal rates were found for the exchange of the first and the second proton.

Evidence for Proton Shuffling in a Thioredoxin-Like Protein during Catalysis

Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol–disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson–Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.

Effects of Proton-Exchange Membrane Fuel-Cell Operating Conditions On Charge Transfer Resistances Measured by Electrochemical Impedance Spectroscopy

Proton-exchange-membrane fuel cells (PEMFC) are highly dependent on operating conditions, such as humidity and temperature. This study employs electrochemical impedance spectroscopy (EIS) to measure the effects of operating parameters on internal proton and electron transport resistance mechanisms in the PEMFC. Current-density experiments have been performed to measure the power production in a 25 cm2 Nafion 117 PEMFC at varying operating conditions. These experiments have shown that low humidity and low temperature contribute to decreased power production. EIS is currently employed to provide a better understanding of the mechanisms involved in power production by calculating the specific resistances at various regions in the PEMFC. Experiments are performed at temperatures ranging from 30 to 50°C, feed humidities from 20 to 98%, and air stoichiometric ratios from 1.33 to 2.67. In all experiments, the hydrogen feed stoichiometric ratio was approximately 4.0. EIS is used to identify which transport steps limit the power production of the PEMFC over these ranges of conditions. The experimental data are analyzed via comparison to equivalent circuit models (ECMs), a technique that uses an electrical circuit to represent the electrochemical and transport properties of the PEMFC. These studies will aid in designing fuel cells that are more tolerant to wide-ranging operating conditions. In addition, optimal operating conditions for PEMFC operation can be identified.

13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s−1 at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN−) was also probed by 13C NMR. A functional C-cluster is not required for 13CN− broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN− exchange appears to monitor only movement of CN− between solution and its binding to and release from CODH.

Determination of target thickness and luminosity from beam energy losses

The repeated passage of a coasting ion beam of a storage ring through a thin target induces a shift in the revolution frequency due to the energy loss in the target. Since the frequency shift is proportional to the beam-target overlap, its measurement offers the possibility of determining the target thickness and hence the corresponding luminosity in an experiment. This effect has been investigated with an internal proton beam of energy 2.65 GeV at the COSY-J\ulich accelerator using the ANKE spectrometer and a hydrogen cluster-jet target. Possible sources of error, especially those arising from the influence of residual gas in the ring, were carefully studied, resulting in an accuracy of better than 5%. The luminosity determined in this way was used, in conjunction with measurements in the ANKE forward detector, to determine the cross section for elastic proton-proton scattering. The result is compared to published data as well as to the predictions of a phase shift solution. The practicability and the limitations of the energy-loss method are discussed.

Mechanisms of photoinduced CαCβ bond breakage in protonated aromatic amino acids

Photoexcitation of protonated aromatic amino acids leads to CαCβ bond breakage among other channels. There are two pathways for the CαCβ bond breakage, one is a slow process (microseconds) that occurs after hydrogen loss from the electronically excited ion, whereas the other is a fast process (nanoseconds). In this paper, a comparative study of the fragmentation of four molecules shows that the presence of the carboxylic acid group is necessary for this fast fragmentation channel to occur. We suggest a mechanism based on light-induced electron transfer from the aromatic ring to the carboxylic acid, followed by a fast internal proton transfer from the ammonium group to the negatively charged carboxylic acid group. The ion formed is a biradical since the aromatic ring is ionized and the carbon of the COOH group has an unpaired electron. Breakage of the weak CαCβ bond gives two even-electron fragments and is expected to quickly occur. The present experimental results together with the ab initio calculations support the interpretation previously proposed.

Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O2 to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O2 reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O2 reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pKa of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O2 reduction, but a decrease in the apparent pKa of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.

Crystal Structure and Raman Studies of dsFP483, a Cyan Fluorescent Protein from Discosoma striata

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins (FPs), we determined the 2.1-Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan class of Anthozoa FPs and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437–440 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the protein-intrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stability. The refined crystal structure of the biological tetramer (space group C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical with that in green fluorescent protein (GFP). To test the roles of specific residues in color modulation, we investigated the optical properties of the H163Q and K70M variants. Although absorbance bands remain broad, peak excitation maxima are red shifted to 455 and 460 nm, emitting cyan light and green light, respectively. To probe chromophore ground-state features, we collected Raman spectra using 752-nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral GFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited-state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.

Determinants of Anion-Proton Coupling in Mammalian Endosomal CLC Proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.

Dynamics of C-phycocyanin in various deuterated trehalose/water environments measured by quasielastic and elastic neutron scattering

The molecular understanding of protein stabilization by the disaccharide trehalose in extreme temperature or hydration conditions is still debated. In the present study, we investigated the role of trehalose on the dynamics of the protein C-phycocyanin (C-PC) by neutron scattering. To single out the motions of C-PC hydrogen (H) atoms in various trehalose/water environments, measurements were performed in deuterated trehalose and heavy water (D2O). We report that trehalose decreases the internal C-PC dynamics, as shown by a reduced diffusion coefficient of protein H atoms. By fitting the Elastic Incoherent Structure Factor—which gives access to the “geometry” of the internal proton motions—with the model of diffusion inside a sphere, we found that the presence of trehalose induces a significantly higher proportion of immobile C-PC hydrogens. We investigated, by elastic neutron scattering, the mean square displacements (MSDs) of deuterated trehalose/D2O-embedded C-PC as a function of temperature in the range of 40–318 K. Between 40 and ∼225 K, harmonic MSDs of C-PC are slightly smaller in samples containing trehalose. Above a transition temperature of ∼225 K, we observed anharmonic motions in all trehalose/water-coated C-PC samples. In the hydrated samples, MSDs are not significantly changed by addition of 15% trehalose but are slightly reduced by 30% trehalose. In opposition, no dynamical transition was detected in dry trehalose-embedded C-PC, whose hydrogen motions remain harmonic up to 318 K. These results suggest that a role of trehalose would be to stabilize proteins by inhibiting some fluctuations at the origin of protein unfolding and denaturation.

Sequence of late molecular events in the activation of rhodopsin

Activation of the G protein-coupled receptor rhodopsin involves both the motion of transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled rhodopsin in solutions of dodecyl maltoside was used so that time-resolved TM6 motion and proton exchange could each be monitored as a function of pH and temperature after an activating light flash. The results reveal that the motion of TM6 is not synchronized with deprotonation of the Schiff base that binds the chromophore to the protein but is an order of magnitude slower at 30 degrees C. However, TM6 motion and the uptake of a proton from solution in the neutral pH range follow the same time course. Importantly, the motion of TM6 is virtually independent of pH, as is Schiff base deprotonation under the conditions used, whereas proton uptake titrates with a pK of 6.5. This finding shows that proton uptake is a consequence rather than a cause of helix motion. Activated rhodopsin binds to and subsequently activates the cognate G protein, transducin. It has been shown that peptides derived from the C terminus of the transducin alpha-subunit mimic in part binding of the intact G protein. These peptides are found to bind to rhodopsin after TM6 movement, resulting in the release of protons. Collectively, the data suggest the following temporal sequence of events involved in activation: (i) internal Schiff base proton transfer; (ii) TM6 movement; and (iii) proton uptake from solution and binding of transducin.

Structural Elements Involved in Proton Translocation by CytochromecOxidase as Revealed by Backbone Amide Hydrogen−Deuterium Exchange of the E286H Mutant

Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa3), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O2 reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains approximately 1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton translocation by CytcO.

Annulenylenes, Annulynes, and Annulenes

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