Proton Positions Research Articles

Atomic Insights into the pH-Induced Activation of Channels and Transporters

Many transmembrane proteins perform functions by utilizing pH-dependent conformational dynamics. However, detailed mechanisms are often elusive due to the challenge in experimentally resolving proton positions and structural details and the inability of conventional molecular dynamics simulations to couple dynamics with proton titration. In this talk I will discuss the application of state-of-the-art continuous constant pH molecular dynamics (CpHMD) to elucidate the conformational activation of Escherichia coli sodium-proton antiporter NhaA and M2 proton channel of influenza virus. As to NhaA, our study revealed the identities of proton carriers and mechanism of sodium-proton exchange. With regards to the M2 channel, simulations revealed the pH-dependent transition from the closed to open channel and provided free energy of channel opening coupled to the stepwise titration of a histidine tetrad. These studies demonstrate CpHMD is a powerful tool to obtain previously unattainable conformational details and thermodynamic information for proton-coupled channels and transporters.

Application of 1H and 27Al magic angle spinning solid state NMR at 60 kHz for studies of Au and Au-Ni catalysts supported on boehmite/alumina

In this work for the first time we show the power of solid state NMR spectroscopy in structural analysis of alumina and catalysts supported on the alumina surface employing very fast (60kHz) magic angle spinning (MAS) technique. In the methodological part we demonstrate that under such MAS condition, cross-polarization (CP) from proton to aluminum is an efficient process when a very weak 27Al RF field is applied. The mechanism of CP transfer and the Hartmann-Hahn (H-H) matching conditions were tested for 27Al RF fields equal to 3.3 and 8.3kHz. It has been found that double quantum (DQ) CP/MAS is the best choice for H-H set with RF =3.3kHz. It has been also proved that the quality of 1H-27Al CP/MAS spectra strongly depends on 27Al carrier offset. Applied to γ-alumina, this method revealed that 1H-27Al CP/MAS at 60kHz is extremely useful for mapping the distribution of hydroxyl groups on the surface. Indeed, the AlV sites, which are not easily detected with Single Pulse Experiment (SPE), are clearly seen when 1H-27Al CP/MAS is applied. Utilizing 2D 1H-27Al CP/MAS HETCOR experiment it was possible to assign the proton positions and to correlate them with aluminum centers. Studies of mono- (Au) and bi- (Au-Ni) metallic catalysts supported on boehmite/alumina carrier employing 1D and 2D HETCOR experiments clearly show that distributions of hydroxyl groups for both systems are dramatically different.

Polar solvent fluctuations drive proton transfer in hydrogen bonded complexes of carboxylic acid with pyridines: NMR, IR and ab initio MD study

We study a series of intermolecular hydrogen-bonded 1 : 1 complexes formed by chloroacetic acid with 19 substituted pyridines and one aliphatic amine dissolved in CD2Cl2 at low temperature by 1H and 13C NMR and FTIR spectroscopy. The hydrogen bond geometries in these complexes vary from molecular (O-HN) to zwitterionic (O-H-N+) ones, while NMR spectra show the formation of short strong hydrogen bonds in intermediate cases. Analysis of C[double bond, length as m-dash]O stretching and asymmetric CO2- stretching bands in FTIR spectra reveal the presence of proton tautomerism. On the basis of these data, we construct the overall proton transfer pathway. In addition to that, we also study by use of ab initio molecular dynamics the complex formed by chloroacetic acid with 2-methylpyridine, surrounded by 71 CD2Cl2 molecules, revealing a dual-maximum distribution of hydrogen bond geometries in solution. The analysis of the calculated trajectory shows that the proton jumps between molecular and zwitterionic forms are indeed driven by dipole-dipole solvent-solute interactions, but the primary cause of the jumps is the formation/breaking of weak CHO bonds from solvent molecules to oxygen atoms of the carboxylate group.

Conformational flexibility and pseudosymmetric aggregation in a betainium salt hydrate

The cation of 3-(trimethylammonium)-benzoic acid exhibits a considerable conformational flexibility connected to the orientation of carboxyl group, coupled to the proton position, and rotations of the trimethylammonium group. This conformational flexibility considerably affects the aggregation of ions and molecules of 3-(trimethylammonium)-benzoic acid iodide hydrate, which crystallizes in a low-symmetry structure with four symmetry-independent formula units (monoclinic space group Pc, Z’ = 4, model I). However this structure approximates the symmetry of space group P21/c in the same unit cell (Z’ = 2, model II), and also even a still more highly symmetric structure of space group P21/c in a half-smaller unit cell (Z’ = 1, model III). The independent formula units are differentiated by the conformations of trimethylammonium groups (ordered in model I, but disordered in models II and III), conformations of the carboxyl groups and positions of OH···O bonded water molecules (ordered in molecules I and II, and disordered in model III), while the bulks of the cations and iodide anions are consistent for all models. While the calorimetric study and varied-temperature structural determinations exclude phase transitions of the highly pseudosymmetric model I. This pseudosymmetry is independent of temperature between 100 K and the m.p. at 463 K, and illustrates subtle structural effects leading complex molecular aggregation in the crystal. Theoretical computations confirm the low energy differences between the conformers.Graphical abstractᅟ

Use of Quantum Theory of Atoms in Molecules in the Search for Appropriate Hydrogen Atom Locations in X-ray Diffraction Based Studies

A novel approach for the determination of hydrogen positions in studies based on X-ray diffraction is presented. The comparison of proton positions with their corresponding electron density maxima has been performed by means of the Quantum Theory of Atoms in Molecules. The observed differences mainly depend on the type and electronegativity of the atom covalently bonded to the hydrogen, changing in the range of Δr = 0.004 ÷ 0.032 A, while being simultaneously independent from the involvement of atom in the hydrogen bonding. It is postulated that the Δr correction should be taken into account in electron density based studies. In theoretical single point calculations (sp) based on X-ray diffraction analysis, an elongation of hydrogen covalent bonds with Δr corrections instead of neutronographic values is proposed. This procedure utilized in this work for the analysis of intramolecular O–H···O hydrogen bonding properties leads to good agreement between quantum theoretical results for the optimized structure...

Assessment of the Presence and Strength of H-Bonds by Means of Corrected NMR

The downfield shift of the NMR signal of the bridging proton in a H-bond (HB) is composed of two elements. The formation of the HB causes charge transfer and polarization that lead to a deshielding. A second factor is the mere presence of the proton-accepting group, whose electron density and response to an external magnetic field induce effects at the position of the bridging proton, exclusive of any H-bonding phenomenon. This second positional shielding must be subtracted from the full observed shift in order to assess the deshielding of the proton caused purely by HB formation. This concept is applied to a number of H-bonded systems, both intramolecular and intermolecular. When the positional shielding is removed, the remaining chemical shift is in much better coincidence with other measures of HB strength.

Conformational Activation of a Transmembrane Proton Channel from Constant pH Molecular Dynamics.

Proton-coupled transmembrane proteins play important roles in human health and diseases; however, detailed mechanisms are often elusive. Experimentally resolving proton positions and structural details is challenging, and conventional molecular dynamics simulations are performed with preassigned and fixed protonation states. To address this challenge, here we illustrate the use of the state-of-the-art continuous constant pH molecular dynamics (CpHMD) to directly describe the activation of the M2 channel of influenza virus, for which abundant experimental data are available. Starting from the closed crystal structure, simulation reveals a pH-dependent conformational switch to an activated state that resembles the open crystal structure. Importantly, simulation affords the free energy of channel opening coupled to the titration of a histidine tetrad, thereby providing a thermodynamic mechanism for M2 activation, that is consistent with NMR data and resolves the controversy with crystal structures obtained at different pH values. This work illustrates the utility of CpHMD in offering previously unattainable conformational details and thermodynamic information for proton-coupled transmembrane channels and transporters.

Can low-barrier hydrogen bond exist in systems with second row elements? An ab initio path integral molecular dynamics study for deprotonated hydrogen sulfide dimer

Nuclear quantum effect and thermal effect on deprotonated hydrogen sulfide dimer anion $${\text{H}}_{3} {\text{S}}_{2}^{-}$$ , composed of a second row element, are widely explored by ab initio on-the-fly path integral molecular dynamics simulation. At low temperature, the hydrogen-bonded proton tends to be diffusively located at the central position between two sulfur atoms, which is the typical characteristic feature of so-called low-barrier hydrogen bond (LBHB). This is the first case of the LBHB systems composed of the second row elements, although the hydrogen-bonded distance in $${\text{H}}_{3} {\text{S}}_{2}^{-}$$ (over 3.4 A) is much longer than the previously reported LBHB composed of first row elements (<2.5 A). At high temperature, the distance between two sulfur atoms is longer than that at low temperature, and the hydrogen-bonded proton localizes to each sulfur atom. Similar tendency is obtained in the deuterated $${\text{D}}_{3} {\text{S}}_{2}^{-}$$ species at all temperature. Analyzing the relationship between the position of the hydrogen-bonded proton and the quantum fluctuation effect of the proton, we elucidate that the LBHB is induced by the quantum tunneling at low temperature, while such trend becomes weak and the character of LBHB vanishes at room temperature for $${\text{H}}_{3} {\text{S}}_{2}^{-}$$ .

Proton transfer pathways in an aspartate-water cluster sampled by a network of discrete states

Proton transfer reactions are complex transitions due to the size and flexibility of the hydrogen-bonded networks along which the protons may “hop”. The combination of molecular dynamics based sampling of water positions and orientations with direct sampling of proton positions is an efficient way to capture the interplay of these degrees of freedom in a transition network. The energetically most favourable pathway in the proton transfer network computed for an aspartate-water cluster shows the pre-orientation of water molecules and aspartate side chains to be a pre-requisite for the subsequent concerted proton transfer to the product state.

Structure Elucidation of Mixed-Linker Zeolitic Imidazolate Frameworks by Solid-State (1)H CRAMPS NMR Spectroscopy and Computational Modeling.

Mixed-linker zeolitic imidazolate frameworks (ZIFs) are nanoporous materials that exhibit continuous and controllable tunability of properties like effective pore size, hydrophobicity, and organophilicity. The structure of mixed-linker ZIFs has been studied on macroscopic scales using gravimetric and spectroscopic techniques. However, it has so far not been possible to obtain information on unit-cell-level linker distribution, an understanding of which is key to predicting and controlling their adsorption and diffusion properties. We demonstrate the use of (1)H combined rotation and multiple pulse spectroscopy (CRAMPS) NMR spin exchange measurements in combination with computational modeling to elucidate potential structures of mixed-linker ZIFs, particularly the ZIF 8-90 series. All of the compositions studied have structures that have linkers mixed at a unit-cell-level as opposed to separated or highly clustered phases within the same crystal. Direct experimental observations of linker mixing were accomplished by measuring the proton spin exchange behavior between functional groups on the linkers. The data were then fitted to a kinetic spin exchange model using proton positions from candidate mixed-linker ZIF structures that were generated computationally using the short-range order (SRO) parameter as a measure of the ordering, clustering, or randomization of the linkers. The present method offers the advantages of sensitivity without requiring isotope enrichment, a straightforward NMR pulse sequence, and an analysis framework that allows one to relate spin diffusion behavior to proposed atomic positions. We find that structures close to equimolar composition of the two linkers show a greater tendency for linker clustering than what would be predicted based on random models. Using computational modeling we have also shown how the window-type distribution in experimentally synthesized mixed-linker ZIF-8-90 materials varies as a function of their composition. The structural information thus obtained can be further used for predicting, screening, or understanding the tunable adsorption and diffusion behavior of mixed-linker ZIFs, for which the knowledge of linker distributions in the framework is expected to be important.

SU‐F‐T‐169: A Periodic Quality Assurance Program for a Spot‐Scanning Proton Treatment Facility

Purpose:To develop daily and monthly quality assurance (QA) programs in support of a new spot‐scanning proton treatment facility using a combination of commercial and custom equipment and software. Emphasis was placed on efficiency and evaluation of key quality parameters.Methods:The daily QA program was developed to test output, spot size and position, proton beam energy, and image guidance using the Sun Nuclear Corporation rf‐DQA™3 device and Atlas QA software. The program utilizes standard Atlas linear accelerator tests repurposed for proton measurements and a custom jig for indexing the device to the treatment couch. The monthly QA program was designed to test mechanical performance, image quality, radiation quality, isocenter coincidence, and safety features. Many of these tests are similar to linear accelerator QA counterparts, but many require customized test design and equipment. Coincidence of imaging, laser marker, mechanical, and radiation isocenters, for instance, is verified using a custom film‐based device devised and manufactured at our facility. Proton spot size and position as a function of energy are verified using a custom spot pattern incident on film and analysis software developed in‐house. More details concerning the equipment and software developed for monthly QA are included in the supporting document. Thresholds for daily and monthly tests were established via perturbation analysis, early experience, and/or proton system specifications and associated acceptance test results.Results:The periodic QA program described here has been in effect for approximately 9 months and has proven efficient and sensitive to sub‐clinical variations in treatment delivery characteristics.Conclusion:Tools and professional guidelines for periodic proton system QA are not as well developed as their photon and electron counterparts. The program described here efficiently evaluates key quality parameters and, while specific to the needs of our facility, could be readily adapted to other proton centers.

Synthesis and Characterization of New 3-(4-Arylpiperazin-1-yl)-2-hydroxypropyl 4-Propoxybenzoates and Their Hydrochloride Salts.

Five new 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-propoxybenzoates were designed and synthesized as potential dual antihypertensive agents. The compounds were prepared as free bases and subsequently transformed to hydrochloride salts. The position of protonation of nitrogen atoms in the piperazine ring of hydrochloride salts was determined by means of 13C-CP/MAS and 15N-CP/MAS NMR and IR spectroscopy. Using these solid-state analytical techniques, it was found that both nitrogen atoms were protonated when excess hydrogen chloride was used for preparation of salts. On the other hand, when the equimolar amount of hydrogen chloride was used, piperazine nitrogen substituted by aryl was protonated.

Evidence of Polaron Excitations in Low Temperature Raman Spectra of Oxalic Acid Dihydrate.

Low temperature Raman spectra of oxalic acid dihydrate (8-300 K) for both the polycrystalline and single crystal phase show strong variation with temperature in the interval from 1200 to 2000 cm(-1). Previous low temperature diffraction studies all confirmed the stability of the crystal P21/n phase with no indications of any phase transition, reporting the existence of a strong hydrogen bond between the oxalic acid and a water molecule. A new group of Raman bands in the 1200-1300 cm(-1) interval below 90 K is observed, caused by possible loss of the center of inversion. This in turn could originate either due to disorder in hydroxyl proton positions or due to proton transfer from carboxylic group to water molecule. The hypothesis of proton transfer is further supported by the emergence of new bands centered at 1600 and 1813 cm(-1), which can be explained with vibrations of H3O(+) ions. The broad band at 1600 cm(-1) looses intensity, while the band at 1813 cm(-1) gains intensity on cooling. The agreement between quantum calculations of vibrational spectra and experimentally observed Raman bands of hydronium ions in oxalic acid sesquihydrate crystal corroborates this hypothesis.

Crystallographic Studies of Human Acetylcholinesterase Reactivation by Oximes: Towards a Neutron Structure

Human acetylcholinesterase (hAChE) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the enzyme's active site leading to a life‐threatening increase of acetylcholine concentration. Exposure to OPs is fatal if not treated with antidotes. However, the current generation of antidotes is not highly efficient, and the rates of reactivation are far slower than the catalytic rate of acetylcholine hydrolysis.Insights into the molecular structures of Torpedo (1) and mammalian AChEs (2) as well as other cholinesterases reveal the underlying limitations in enhancing reactivation rates but are limited in their ability to resolve positions of protons and hydrogens important for understanding of proton transfer in reactivation of organophosphate inhibited cholinesterases. The only experimental method that can provide this information is macromolecular neutron crystallography. In combination with X‐ray crystallography it is capable of providing insights into the active sites of enzymes with the atomic resolution.In preparation for the neutron diffraction studies we have obtained several new high resolution X‐ray structures of hAChE at room and low temperatures in the apo form and in complex with oxime reactivators and other ligands. The enzyme crystallizes in a novel crystal form with the unit cell amenable to neutron crystallography (space group P31, a=b=124.3, c=129.1 Å). Detailed analysis of the reactivators and ligand interactions at the enzyme active will be presented.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.

A DFT-Elucidated Comparison of the Solution-Phase and SAM Electrochemical Properties of Short-Chain Mercaptoalkylferrocenes: Synthetic and Spectroscopic Aspects, and the Structure of Fc-CH2CH2-S-S-CH2CH2-Fc.

Facile synthetic procedures to synthesize a series of difficult-to-obtain mercaptoalkylferrocenes, namely, Fc(CH2)nSH, where n = 1 (1), 2 (2), 3 (3), or 4 (4) and Fc = Fe(η(5)-C5H5)(η(5)-C5H4), are reported. Dimerization of 1-4 to the corresponding disulfides 19-22 was observed in air. Dimer 20 (Z = 2) crystallized in the triclinic space group P1̅. Dimers 20-22 could be reduced back to the original Fc(CH2)nSH derivatives with LiAlH4 in refluxing tetrahydrofuran. Density functional theory (DFT) calculations showed that the highest occupied molecular orbital of 1-4 lies exclusively on the ferrocenyl group implying that the electrochemical oxidation observed at ca. -15 < Epa < 76 mV versus FcH/FcH(+) involves exclusively an Fe(II) to Fe(III) process. Further DFT calculations showed this one-electron oxidation is followed by proton loss on the thiol group to generate a radical, Fc(CH2)nS(•), with spin density mainly located on the sulfur. Rapid exothermic dimerization leads to the observed dimers, Fc(CH2)n-S-S-(CH 2)nFc. Reduction of the ferrocenium groups on the dimer occurs at potentials that still showed the ferrocenyl group ΔE = Epa,monomer - Epc,dimer ≤ 78 mV, indicating that the redox properties of the ferrocenyl group on the mercaptans are very similar to those of the dimer. (1)H NMR measurements showed that, like ferrocenyl oxidation, the resonance position of the sulfhydryl proton, SH, and others, are dependent on -(CH2)n- chain length. Self-assembled monolayers (SAMs) on gold were generated to investigate the electrochemical behavior of 1-4 in the absence of diffusion. Under these conditions, ΔE approached 0 mV for the longer chain derivatives at slow scan rates. The surface-bound ferrocenyl group of the metal-thioether, Fc(CH2)n -S-Au, is oxidized at approximately equal potentials as the equivalent CH2Cl2-dissolved ferrocenyl species 1-4. Surface coverage by the SAMs is dependent on alkyl chain length with the largest coverage obtained for 4, while the rate of heterogeneous electron transfer between SAM substrate and electrode was the fastest for the shortest chain derivative, Fc-CH2-S-Au.

α-Substituted Bis(octabutoxyphthalocyaninato)Terbium(III) Double-Decker Complexes: Preparation and Study of Protonation by NMR and DFT.

Synthesis of the anionic, α-substituted, bis(phthalocyaninato)Tb(III) complex [Tb(α-obPc)2](-) ([1](-)) (obPc = α-octabutoxyphthalocyaninato) leads to the isolation of its protonated form [1H](0). This complex was characterized by X-ray diffraction (XRD), mass spectroscopy (MS), infrared (IR) and ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy. Crystal structure analysis did not allow localization of the additional proton, which is probably attached to the meso-N atom or isoindole-N atom of the phthalocyaninato ligand. [1H](0) can easily be deprotonated or protonated, giving the corresponding anionic and cationic complexes. The three compounds [1H](0), [1](-), and [1HH](+) were studied by a combination of paramagnetic NMR experiments ((1)H, (13)C, variable-temperature measurements, two-dimensional nuclear magnetic resonance and DFT calculations (done on Y(III) analogues with octamethoxyphthalocyaninato ligands), for the purpose of elucidating the positions of the acidic protons and for understanding the structural changes of the coordination environment of the Tb ion induced by protonation.

From crystalline to amorphous calcium pyrophosphates: A solid state Nuclear Magnetic Resonance perspective

Hydrated calcium pyrophosphates (CPP, Ca2P2O7·nH2O) are a fundamental family of materials among osteoarticular pathologic calcifications. In this contribution, a comprehensive multinuclear NMR (Nuclear Magnetic Resonance) study of four crystalline and two amorphous phases of this family is presented. 1H, 31P and 43Ca MAS (Magic Angle Spinning) NMR spectra were recorded, leading to informative fingerprints characterizing each compound. In particular, different 1H and 43Ca solid state NMR signatures were observed for the amorphous phases, depending on the synthetic procedure used. The NMR parameters of the crystalline phases were determined using the GIPAW (Gauge Including Projected Augmented Wave) DFT approach, based on first-principles calculations. In some cases, relaxed structures were found to improve the agreement between experimental and calculated values, demonstrating the importance of proton positions and pyrophosphate local geometry in this particular NMR crystallography approach. Such calculations serve as a basis for the future ab initio modeling of the amorphous CPP phases. Statement of significanceThe general concept of NMR crystallography is applied to the detailed study of calcium pyrophosphates (CPP), whether hydrated or not, and whether crystalline or amorphous. CPP are a fundamental family of materials among osteoarticular pathologic calcifications. Their prevalence increases with age, impacting on 17.5% of the population after the age of 80. They are frequently involved or associated with acute articular arthritis such as pseudogout. Current treatments are mainly directed at relieving the symptoms of joint inflammation but not at inhibiting CPP formation nor at dissolving these crystals. The combination of advanced NMR techniques, modeling and DFT based calculation of NMR parameters allows new original insights in the detailed structural description of this important class of biomaterials.

COMPREHENSIVE RADIATION DOSE MEASUREMENTS AND MONTE CARLO SIMULATION FOR THE 7Li(p,n) ACCELERATOR NEUTRON FIELD.

In order to investigate the radiation dose dependence on the incident proton energy, neutron and gamma-ray doses were measured using a tissue-equivalent proportional counter in the proton energy range of 1.95-2.50 MeV for the McMaster 7Li(p,n) neutron facility. Microdosimetric spectra were collected, and absorbed doses were determined at various positions inside the irradiation cavity, along the lateral axis and outside the shield to find out the spatial distributions of neutron and gamma-ray doses for each proton energy. In parallel with the absorbed dose measurements, MCNP Monte Carlo simulations were carried out and neutron fluence spectra were computed at various positions, which enabled determination of the neutron weighting factors. It was found that neutrons make a substantially dominant contribution to the total equivalent dose for most proton energies and positions. The effective dose for a human subject increased from 0.058 to 1.306 μSv μA-1 min-1 with the increase of proton energy from 1.95 to 2.5 MeV. It is expected that the reported data will be useful for 7Li(p,n) accelerator neutron users.

Protonation states and catalysis: Molecular dynamics studies of intermediates in tryptophan synthase.

The importance of protonation states and proton transfer in pyridoxal 5'-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global protein dynamics during the catalytic cycle. In general, proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate, such as the phosphoryl group, pyridine nitrogen, phenolic oxygen and carboxyl group, of each PLP-bound intermediate play a crucial role in constructing an appropriate molecular interface with TRPS. In particular, the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition, remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes.

DFT Study of Proton Transfer in Methyl Urocanate and Butyl Urocanate

SummaryDevelopment of proton conductivity is necessary to improve polymer electrolyte efficiency of proton exchange membrane fuel cell (PEMFC) which is alternative environmentally friendly resource. Modification of polymer electrolyte has been employed using water free polymer instead of hydrated polymer membrane to disregard water loss problem at high temperature. Heterocycles such as imidazole and benzimidazole derivatives was considered for anhydrous membrane modification to encourage proton transfer in polymer electrolyte membrane. Alkyl urocanate was interested as a vehicle for proton transfer between side chains of polymer membrane. In this work, investigation of blending of butyl urocanate in polymer membrane to assist the proton transfer ability was carried out. Energy barrier of proton movement between alkyl urocanate was calculated. The preferred orientation of bimolecular alkyl urocanaetes was initiated by crystallographic data. The energies of various proton position in between neighbouring molecules were calculated to determine the barrier of proton transfer. The density function theory (DFT) with generalized gradient approximation (GGA) and BLYP function was used for calculation. The energy barrier of butyl urocanate was found lower than methyl urocanate corresponding to the previous experimental proton conductivity data.