Grotthuss Proton Research Articles

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.

The Molecular Mechanisms of Neural Flow Coupling: A New Concept.

ABSTRACTThe phenomenon known as neural flow coupling (NFC) occurs at the capillary level where there are no known pressure controlling structures. Recent developments in advanced magnetic resonance imaging technologies have made possible in vivo direct investigations of water physiology that have shed new insight on the water dynamics of the cortical pericapillary space and their complex functionality in relation to NFC. Neural activities initiate a chain of events that ultimately affect NFC. First, neural activities generate extracellular acidification. Extracellular acidosis in turn produces inhibition of aquaporin‐4 (AQP‐4) located at the end feet of pericapillary astrocytes, the water channel which regulates water influx into the pericapillary space and, hence, interstitial flow. Reduction of pericapillary water pressure results in a negative balance between pericapillary and intraluminal capillary pressure, allowing for capillary caliber expansion. Proton permeability through the tight junctions of the blood brain barrier is significantly high owing to the Grotthuss proton “tunneling” mechanism and, therefore, carbonic anhydrase (CA) type IV (CA‐IV) anchored to the luminal surface of brain capillaries functions as scavenger of extracellular protons. CA‐IV inhibition by acetazolamide or carbon dioxide results in the accumulation of extracellular protons, causing AQP‐4 inhibition and a secondary increase in rCBF.

Differentiating Grotthuss proton conduction mechanisms by nuclear magnetic resonance spectroscopic analysis of frozen samples.

Available methods to analyze proton conduction mechanisms cannot distinguish between two proton-conduction processes derived from the Grotthuss mechanism. The two mechanistic variations involve structural diffusion, for which water movement is indispensable, and the recently proposed "packed-acid mechanism," which involves the conduction of protons without the movement of water and is typically observed in materials consisting of highly concentrated (packed) acids. The latter mechanism could improve proton conductivity under low humidity conditions, which is desirable for polymer electrolyte fuel cells. We proposed a method with which to confirm quantitatively the packed-acid mechanism by combining (2)H and (17)O solid-state magic-angle-spinning nuclear magnetic resonance (MAS-NMR) measurement and (1)H pulsed-field gradient (PFG)-NMR analysis. In particular, the measurements were performed below the water-freezing temperature to prevent water movement, as confirmed by the (17)O-MAS-NMR spectra. Even without water movement, the high mobility of protons through short- and long-range proton conduction was observed by using (2)H-MAS-NMR and (1)H-PFG-NMR techniques, respectively, in the composite of zirconium sulfophenylphosphonate and sulfonated poly(arylene ether sulfone) (ZrSPP-SPES), which is a material composed of highly concentrated acids. Such behavior contrasts with that of a material conducting protons through structural diffusion or vehicle mechanisms (SPES), in which the peaks in both (2)H and (17)O NMR spectra diminished below water-freezing temperature. The activation energies of short-range proton movement are calculated to be 2.1 and 5.1 kJ/mol for ZrSPP-SPES and SPES, respectively, which indicate that proton conduction in ZrSPP-SPES is facilitated by the packed-acid mechanism. Furthermore, on the basis of the mean-square displacement using the diffusivity coefficient below water-freezing temperature, it was demonstrated that long-range proton movement, of the order of 1.3 μm, can take place in the packed-acid mechanism in ZrSPP-SPES.

Selective Analysis of Grotthuss Proton Diffusion in Polymer Electrolyte Membranes at a Freezing Temperature Region: A 2H and 17O Solid-State NMR Investigation

Molecular-level understanding of dynamic behaviors of complex mass transports –especially proton and water- in polymer electrolytes is essential to design better membrane materials used for polymer electrolyte fuel cells (PEFCs). NMR is one of the suitable experimental techniques for analyzing such molecular dynamics, since both complex static structures and dynamical motions of molecules can be tracked by high-resolution NMR spectra and a pulsed field gradient NMR (PFG-NMR). Widely used a pulsed field gradient NMR method, however, cannot clearly distinguish vehicle and grotthuss mechanisms. To overcome this drawback, we applied a combination method of 2H, 17O MAS NMR and 1H PFG NMR measurement. We selectively observed grotthuss mechanism in the absence of a vehicle mechanism in a water-freezing temperature region.In order to demonstrate advantage of this method, we applied this to sulphonated poly (arylene ether sulphone) (SPES) and a composite of zirconium sulphophenylphosphonate (ZrSPP) and SPES using nano capping technique (Capping-ZrSPP-SPES), in which enhanced grottuss mechanism is expected by controlled configuration of proton functional groups. [1, 2] 2H and 17O labeled water was used in order to distinguish signals from non-exchangeable protons in SPES, and because natural abundance 17O (0.04%) NMR signals of water in membrane cannot be detected. Incubating the composites in 2H labeled water at 100% RH for three days resulted in the exchange of the protons of sulfonic acid and adsorbed water, and thus the 2H MAS NMR spectrum included only the signal of the mobile proton. Similarly, for 17O MAS NMR analysis, the composite was incubated in 17O labeled water at 100% RH for three days. After reaching equilibrium, air tight sample tubes were sealed to avoid change of the water content during NMR measurements. 2H and 17O MAS NMR measurements were carried out on Agilent NMR systems 400WB at 9.4 T. A 4 mm diameter air tight rotor was used for sample spin at 12 kHz. 2H and 17O 1D-NMR spectra were obtained by using a single pulse sequence. Sample temperature was calibrated by measuring the 79Br signal of KBr powder under the same measurement condisions as described elsewhere. [3] 1H PFG NMR measurements were carried out on JNM LA-400 at 9.4 T. NMR spectrometer equipped with maximum gradient strength of 12 T/m. The diffusion coefficient (D) was measured by using a pulsed-field-gradient stimulated-echo pulse sequence. [4, 5]Fig. 1 shows the temperature variations of 2H and 17O NMR spectra of SPES, respectively, at 47, -15 and -39 °C. 2H and 17O NMR singnals of SPES are broadened as temperature decrease. At -39 °C, 2H and 17O NMR signals are significantly broadened. This indicates that water is freezing at -39 °C and proton in SPES cannot move.On the other hand, the temperature variation of 2H NMR signal from Capping-ZrSPP-SPES showed different behaviors. As shown in Fig. 2 (b), 2H resonance peak is still sharp in the water-freezing temperature, suggesting that the protons in the Capping-ZrSPP–SPES retained their mobility even at -39 °C. These results indicate that protons in the Capping-ZrSPP–SPES move independently like a grotthuss mechamisum in freezing temperature region. More detailed and 1H PFG NMR results will be presented at the Meeting.

Anomalous Surface Diffusion of Protons on Lipid Membranes

The cellular energy machinery depends on the presence and properties of protons at or in the vicinity of lipid membranes. To asses the energetics and mobility of a proton near a membrane, we simulated an excess proton near a solvated DMPC bilayer at 323 K, using a recently developed method to include the Grotthuss proton shuttling mechanism in classical molecular dynamics simulations. We obtained a proton surface affinity of −13.0 ± 0.5 kJ mol−1. The proton interacted strongly with both lipid headgroup and linker carbonyl oxygens. Furthermore, the surface diffusion of the proton was anomalous, with a subdiffusive regime over the first few nanoseconds, followed by a superdiffusive regime. The time- and distance dependence of the proton surface diffusion coefficient within these regimes may also resolve discrepancies between previously reported diffusion coefficients. Our simulations show that the proton anomalous surface diffusion originates from restricted diffusion in two different surface-bound states, interrupted by the occasional bulk-mediated long-range surface diffusion. Although only a DMPC membrane was considered in this work, we speculate that the restrictive character of the on-surface diffusion is highly sensitive to the specific membrane conditions, which can alter the relative contributions of the surface and bulk pathways to the overall diffusion process. Finally, we discuss the implications of our findings for the energy machinery.

Charge transport in poly-imidazole membranes: a fresh appraisal of the Grotthuss mechanism

A detailed theoretical investigation of the charge transport mechanism in poly(4-vinyl-imidazole) (P4VI), the parent polymer of a series of N-heterocyclic-based membranes used as an electrolyte in proton exchange membrane fuel cells, is presented. In particular, Density Functional Theory (DFT) results obtained for small model systems (protonated imidazole dimers and trimers) suggest that the commonly accepted conduction mechanism, based on a sequential proton transfer between imidazole moieties, could be impeded by the geometrical constraints imposed by the polymeric backbone. Indeed only one kind of proton transfer reaction is energetically allowed between adjacent imidazoles, so that a rotation of the protonated imidazole is required for a second proton transfer. Molecular dynamics simulations on a larger model (15 oligomers with an excess proton) show that the rotation of the imidazole carrying the excess proton is a soft large amplitude motion. These results allow us to propose a new proton conduction mechanism in P4VI, where a frustrated rotation of the protonated imidazole before each proton transfer reaction represents the rate-limiting step. Furthermore, in contrast with the Grotthuss proton transport mechanism in water, our results indicate that here it is the same proton which could be successively transferred. From a chemical point of view, these new insights into the mechanism are relevant for a rational design of modified azole-based systems for Proton Exchange Membrane Fuel Cells.

Proton Solvation and Transport in Hydrated Nafion

Proton solvation properties and transport mechanisms have been studied in hydrated Nafion using the self-consistent multistate empirical valence bond (SCI-MS-EVB) method that includes the effects excess proton charge defect delocalization and Grotthuss proton hopping. It was found that sulfonate groups influence excess proton solvation, as well as the proton hydration structure, by stabilizing a more Zundel-like (H(5)O(2)(+)) structure in their first solvation shells. Hydrate proton-related hydrogen bond networks were observed to be more stable than networks with water alone. Diffusion rates, Arrhenius activation energies, and transport pathways were calculated and analyzed to characterize the nature of the proton transport. Diffusion rate analysis suggests that a proton-hopping mechanism dominates the proton transport for the studied water loading levels and that there is a clear degree of anticorrelation with the vehicular transport. The activation energy drops quickly with an increasing water content when the water loading level is smaller than ∼10 H(2)O/SO(3)(-), which is consistent with experimental observations. The sulfonate groups were also found to affect the proton hopping directions. The temperature and water content effects on the proton transport pathways were also investigated.

A fundamental study of the transport properties of aqueous superacid solutions.

An extensive investigation of the transport properties of aqueous acid solutions was undertaken. The acids studied were trifluoromethanesulfonic (CF(3)SO(3)H), bis(trifluoromethanesulfonyl)imide [(CF(3)SO(2))(2)NH], and para-toluenesulfonic (CH(3)C(6)H(4)SO(3)H), of which the first two are considered superacids. NMR measurements of self-diffusion coefficients (D), spin-lattice relaxation times (T(1)), and chemical shifts, in addition to ionic conductivity (sigma), viscosity (eta), and density measurements, were performed at 30 degrees C over the concentration range of 2-112 water to acid molecules. Results showed broad maxima in sigma for all three acids in the concentration range of 12-20 water to acid molecules. This coincided with minima in anion Ds and is attributed to a local molecular ordering, reduced solution dielectric permittivity, and increased ionic interactions. The location of the maxima in sigma correlates with what is observed for hydrated sulfonated perfluoropolymers such as Nafion, which gives a maximum in ionic transport when the ratio of water to acid molecules is about 15-20. Of the three acids, bis(trifluoromethanesulfonyl)imide was found to be the least dependent on hydration level. The occurrence of the anticorrelation between the ionic conductivity maximum and the anion self-diffusion minimum supports excess proton mobility in this region and may offer additional information on the strength of hydrogen bonding in aqueous media as well as on the role of high acid concentration in the Grotthuss proton transport mechanism.

An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1.

Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton 'hopping' in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.

Determinant of Cation Blocking Behavior in Aquaporin-1

The most extensively studied member of the aquaporin family, wildtype (WT) aquaporin-1 (AQP1), has been shown to effectively block cations from permeation across the cell membrane, thus maintaining the osmotic pressure of the cell. Recent experiments have suggested the essential role that the Selectivity Filter (SF) region of the channel plays in blocking cation flux. In the present study, the potential of mean forces for permeation of sodium cation (Na+), potassium cation (K+), and classical hydronium cation (H3O+) (without the possibility of Grotthuss proton shuttling) are characterized for a series of AQP1 mutants. The free energy barriers for conducting hydrated excess protons (H+), which diffuse via the Grotthuss mechanism, are characterized as well, by utilizing the multi-state empirical valence bond (MS-EVB) method. The maximum cation conductance is calculated using the Poisson-Nerst-Planck theory. The present study reveals the key role of the SF domain in cation gating and provides insight into the subtle mechanism of proton permeation mutants of the AQP1 channel.

Proton Transport Through Channels: Insights and Surprises from Molecular Simulation

The behavior of hydrated excess protons passing into and through transmembrane proton channels will be presented, based on the results of a novel multi-scale computer simulation methodology. The unique electrostatics related to the dynamic delocalization of the excess proton charge defect will be elaborated, as well as its effect on the channel proton transport and selectivity properties. The often opposing and asymptotic viewpoints related to electrostatics on one hand and Grotthuss proton shuttling on the other will be reconciled and unified into a single conceptual framework. Specific simulation results will be given for various channel systems, including the M2 channel of influenza A, proton selective mutant aquaporin-1 channels, the ClC Cl-/H+ antiporter, and models of the Hv1 voltage gated proton channel. Comparison to experimental results will be discussed where possible.

Framework Models of Ion Permeation Through Membrane Channels and the Generalized King–Altman Method

A modern approach to studying the detailed dynamics of biomolecules is to simulate them on computers. Framework models have been developed to incorporate information from these simulations in order to calculate properties of the biomolecules on much longer time scales than can be achieved by the simulations. They also provide a simple way to think about the simulated dynamics. This article develops a method for the solution of framework models, which generalizes the King-Altman method of enzyme kinetics. The generalized method is used to construct solutions of two framework models which have been introduced previously, the single-particle and Grotthuss (proton conduction) models. The solution of the Grotthuss model is greatly simplified in comparison with direct integration. In addition, a new framework model is introduced, generalizing the shaking stack model of ion conduction through the potassium channel.

Computer Simulation of Proton Solvation and Transport in Aqueous and Biomolecular Systems

The excess proton in aqueous media is critical to many aspects of chemistry, biology, and materials science. This species is at the heart of the most elementary of chemical (e.g., acid-base) and biological (e.g., bioenergetics) concepts, yet to this day, it remains mysterious, surprising, and often misunderstood. In this Account, our efforts to describe excess proton solvation and transport through computer modeling and simulation will be described. Results will be summarized for several important systems, as obtained from the multistate empirical valence bond (MS-EVB) approach, which allows for the explicit treatment of (Grotthuss) proton shuttling and charge delocalization.

Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy

Much progress has been made in our understanding of water molecule reactions on surfaces, proton solvation in gas-phase water clusters and proton transfer through liquids. Compared with our advanced understanding of these physico-chemical systems, much less is known about individual water molecules and their cooperative behaviour in heterogeneous proteins during enzymatic reactions. Here we use time-resolved Fourier transform infrared spectroscopy (trFTIR) and in situ H2(18)O/H2(16)O exchange FTIR to determine how the membrane protein bacteriorhodopsin uses the interplay among strongly hydrogen-bonded water molecules, a water molecule with a dangling hydroxyl group and a protonated water cluster to transfer protons. The precise arrangement of water molecules in the protein matrix results in a controlled Grotthuss proton transfer, in contrast to the random proton migration that occurs in liquid water. Our findings support the emerging paradigm that intraprotein water molecules are as essential for biological functions as amino acids.

Computer simulation of explicit proton translocation in cytochrome c oxidase: the D-pathway.

Proton translocation in the D-pathway of cytochrome c oxidase has been studied by a combination of classical molecular dynamics and the multistate empirical valence bond methodology. This approach allows for explicit Grotthuss proton hopping between water molecules. According to mutagenesis experiments, the role of proton donor/acceptor along the D-pathway is carried by the highly conserved residue Glu-242. The present multistate empirical valence bond simulations indicate that the protonation/deprotonation state of Glu-242 is strongly coupled to the distance of proton propagation in the D-pathway. The proton was seen to travel the full length of the D-pathway when Glu-242 was deprotonated; however, it was trapped halfway along the path when Glu-242 was protonated. Further investigation in terms of both proton dynamical properties and free energy calculations for the pathway of proton transport provides evidence for a two-step proton transport mechanism in the D-pathway.

Erratum to: H+-catalyzed symmetric proton exchange in neat liquids with a network of N–H⋯N and O–H⋯O hydrogen bonds and molecular mechanism of Grotthuss proton migration [Chem. Phys. 282 (2002) 51–61

Erratum to: H+-catalyzed symmetric proton exchange in neat liquids with a network of N–H⋯N and O–H⋯O hydrogen bonds and molecular mechanism of Grotthuss proton migration [Chem. Phys. 282 (2002) 51–61

H +-catalyzed symmetric proton exchange in neat liquids with a network of N–H⋯N and O–H⋯O hydrogen bonds and molecular mechanism of Grotthuss proton migration

Proton exchange rates provide direct information about the mean lifetime τ H + and Grotthuss mobility 1/ τ H + of solvated protons in liquids. Here we will analyze our own data of NMR studies on liquid hydrazine and its methyl derivatives as well as the available data on liquid NH 3, methanol, and water with special emphasis on relationship between 1/ τ H + and (a) orientational mobility of molecules, (b) structure of hydrogen-bonded liquids, and (c) structure of proton solvates. Variation in 1/ τ H +, different temperature behavior, and unexpectedly high proton mobility in some hydrazines can be rationalized in terms of the so-called structural diffusion, that is, diffusion of structural defects formed by excess protons in hydrogen-bonded liquids. Also discussed is the effect of hydrophobic substituents.