Role of water structure in alkaline water electrolysis.

Herein, with the help of experimental and first-principles density functional theory (DFT)-based studies, we have shown that structural changes in the water coordination in electrolytes having high alkalinity can be a possible reason for the reduced catalytic activity of platinum (Pt) in high pH. Studies with polycrystalline Pt electrodes indicate that electrocatalytic HER activity reduces in terms of high overpotential required, high Tafel slope, and high charge transfer resistances in concentrated aqueous alkaline electrolytes (say 6 M KOH) in comparison to that in low alkaline electrolytes (say 0.1 M KOH), irrespective of the counter cations (Na+, K+, or Rb+) present. The changes in the water structure of bulk electrolytes as well as that in electrode-electrolyte interface are studied. The results are compared with DFT-based analysis, and the study can pave new directions in studying the HER process in terms of the water structure near the electrode-electrolyte interface.

Realizing 1,1-Dehydration of Secondary Alcohols to Carbenes: Pyrrolidin-2-ols as a Source of Cyclic (Alkyl)(Amino)Carbenes.

Herein we report secondary pyrrolidin-2-ols as a source of cyclic (alkyl)(amino)carbenes (CAAC) for the synthesis of CAAC-CuI -complexes and cyclic thiones when reacted with CuI -salts and elemental sulfur, respectively, under reductive elimination of water from the carbon(IV)-center. This result demonstrates a convenient and facile access to CAAC-based CuI -salts, which are well known catalysts for different organic transformations. It further establishes secondary alcohols to be a viable source of carbenes-realizing after 185 years Dumas' dream who tried to prepare the parent carbene (CH2 ) by 1,1-dehydration of methanol. Addressed is also the reactivity of water towards CAACs, which proceeds through an oxidative addition of the O-H bond to the carbon(II)-center. This emphasizes the ability of carbon-compounds to mimic the reactivity of transition-metal complexes: reversible oxidative addition and reductive elimination of the O-H bond to/from the C(II)/C(IV)-centre.

Spontaneous and frequent conformational dynamics induced by A A mismatch in d(CAA)·d(TAG) duplex.

Base pair mismatches in DNA can erroneously be incorporated during replication, recombination, etc. Here, the influence of A…A mismatch in the context of 5'CAA·5'TAG sequence is explored using molecular dynamics (MD) simulation, umbrella sampling MD, circular dichroism (CD), microscale thermophoresis (MST) and NMR techniques. MD simulations reveal that the A…A mismatch experiences several transient events such as base flipping, base extrusion, etc. facilitating B-Z junction formation. A…A mismatch may assume such conformational transitions to circumvent the effect of nonisostericity with the flanking canonical base pairs so as to get accommodated in the DNA. CD and 1D proton NMR experiments further reveal that the extent of B-Z junction increases when the number of A…A mismatch in d(CAA)·d(T(A/T)G) increases (1-5). CD titration studies of d(CAA)·d(TAG)n=5 with the hZαADAR1 show the passive binding between the two, wherein, the binding of protein commences with B-Z junction recognition. Umbrella sampling simulation indicates that the mismatch samples anti…+ syn/+ syn…anti, anti…anti & + syn…+ syn glycosyl conformations. The concomitant spontaneous transitions are: a variety of hydrogen bonding patterns, stacking and minor or major groove extrahelical movements (with and without the engagement of hydrogen bonds) involving the mismatch adenines. These transitions frequently happen in anti…anti conformational region compared with the other three regions as revealed from the lifetime of these states. Further, 2D-NOESY experiments indicate that the number of cross-peaks diminishes with the increasing number of A…A mismatches implicating its dynamic nature. The spontaneous extrahelical movement seen in A…A mismatch may be a key pre-trapping event in the mismatch repair due to the accessibility of the base(s) to the sophisticated mismatch repair machinery.

Cryo vs Thermo: Duality of Ethylene Glycol on the Stability of Proteins.

Osmolytes are known to stabilize proteins under stress conditions. Thermal denaturation studies on globular proteins (ß-lactoglobulin, cytochrome c, myoglobin, α-chymotrypsin) in the presence of ethylene glycol (EG), a polyol class of osmolyte, demonstrate a unique property of EG. EG stabilizes proteins against cold denaturation and destabilizes them during heat-induced denaturation. Further, chemical denaturation experiments performed at room temperature and at a sub-zero temperature (-10 °C) show that EG stabilizes the proteins at subzero temperature but destabilizes them at room temperature. The experiments carried out in the presence of glycerol, however, showed that glycerol stabilizes proteins against all of the denaturing conditions. This differential effect has not been reported for any other polyol class of osmolyte and might be specific to EG. Moreover, molecular dynamics simulations of all of the four proteins were carried out at three different temperatures, 240, 300, and 340 K, in the absence and presence of EG (20 and 40%). The results suggest that EG preferably accumulates around the hydrophobic residues and reduces the hydrophobic hydration of the proteins at a low temperature leading to stabilization of the proteins. At 340 K, the preferential hydration of the proteins is significantly reduced and the preferential binding of EG destabilizes the proteins like common denaturants.

α,α'-Diamino-p-quinodimethanes with Three Stable Oxidation States.

Herein, we report the rational design, synthesis, and characterization of α,α'-diamino-substituted-p-quinodimethanes, which are a group of partially substituted p-quinodimethanes. These exhibit two reversible one-electron redox steps and electrochromism in the ultraviolet, visible, and near-infrared regions. We were able to isolate the crystalline compounds of all three oxidation states: neutral, radical cation, and dication. The obtained results not only create the bridge between p-quinodimethane and α,α,α',α'-tetrasubstituted-p-quinodimethane, but also demonstrate the straightforward modular approach for the synthesis of π-conjugated open-shell compounds.

Neutral and anionic phosphate-diesters as molecular templates for the encapsulation of a water dimer.

The study of water clusters in a confined state has received considerable attention. The simplest among such assemblies of water molecules is the water dimer. Stabilizing this unit at ambient conditions in an appropriate host-template, however, has remained a challenge. Herein, we report 2,6-(diphenylmethyl)-4-iso-propyl-phenyl substituted phosphate diesters (both in their neutral and anionic forms) as molecular templates for hosting the water dimer. The robustness of the water dimer within the neutral phosphate-diester ensemble is remarkable. Even when treated with triethylamine the water dimer is not disrupted, while exclusively the P(O)(OH) unit is deprotonated.

Influence of local structural disorders on spectroscopic properties of multi-component CaF2-Bi2O3-P2O5-B2O3 glass ceramics with Cr2O3 as nucleating agent.

Multi-component CaF2-Bi2O3-P2O5-B2O3 glasses doped with different concentrations of Cr2O3 were crystallized through heat treatment. The prepared glass ceramic samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and differential thermal analysis (DTA). Spectroscopic studies viz., optical absorption, Fourier transform infrared (FTIR), Raman and electron paramagnetic resonance (EPR) were carried out. The XRD, SEM and DTA studies indicated that the samples contain different crystalline phases. Results of optical absorption and EPR studies pointed out the gradual conversion of chromium ions from Cr(3+) state to Cr(6+) state with an increase of Cr2O3 content from 0.1 to 0.5 mol%. The results of FTIR, Raman and EPR studies revealed that Cr(6+) ions participate in the glass network in tetrahedral positions and seemed to increase the polymerization of the glass ceramics. The quantitative analysis of results of the spectroscopic studies further indicated that the glasses crystallized with low concentration of Cr2O3 are favourable for solid state laser devices.

Long-lived spin state of a tripeptide in stretched hydrogel.

The longitudinal (T 1), transverse (T 2), and singlet state (T s) relaxation times of the geminal backbone protons (CH2) of L-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D2O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T 2 and T s of the CH2 protons correspondingly decreased (T s/T 2 ~ constant), while the change in T 1 was relatively small. The largest observed T s/T 1 value was 3.3 at 46% w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D2O in (2)H NMR spectra, and residual dipolar splitting of the CH2 resonance in (1)H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T 1, T 2, and T s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T 2 is shorter than T 1 in the presence of non-zero residual dipolar couplings.

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses.

Li(2)O-ZrO(2)-SiO(2):Nd(3+) glasses mixed with Nb(2)O(5), Ta(2)O(5) and La(2)O(3) were prepared. Optical absorption and photoluminescence spectra of these glasses have been recorded at room temperature. The Judd-Ofelt theory was successfully applied to characterize Nd(3+) spectra of all the three glasses. From this theory, various radiative properties like transition probability A, branching ratio ß(r), the radiative lifetime τ(r), for (4)F(3/2) emission level in the spectra of these glasses has been evaluated. The radiative life time for (4)F(3/2) level of Nd(3+) ions has also been measured and quantum efficiencies were estimated. Among the three glasses studied, the La(2)O(3) mixed glass has exhibited the highest quantum efficiency. The reasons for such higher value have been discussed based on the relationship between the structural modifications taking place around the Nd(3+) ions.

15N H/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3.

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide (15)N is used as a probe of solvent exchange. The experiment is performed in 50% H(2)O-50% D(2)O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, (15)N(D), into protonated species, (15)N(H), as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of (15)N(H/D)-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5-2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.

Paramagnetic relaxation enhancements in unfolded proteins: theory and application to drkN SH3 domain.

Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as (1)H(N) is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the (1)H(N) residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between (1)H(N) spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.

Complexity of aromatic ring-flip motions in proteins: Y97 ring dynamics in cytochrome c observed by cross-relaxation suppressed exchange NMR spectroscopy.

Dynamics of large-amplitude conformational motions in proteins are complex and less understood, although these processes are intimately associated with structure, folding, stability, and function of proteins. Here, we use a large set of spectra obtained by cross-relaxation suppressed exchange NMR spectroscopy (EXSY) to study the 180 degrees flipping motion of the Y97 ring of horse ferricytochrome c as a function of near-physiological temperature in the 288-308 K range. With rising temperature, the ring-flip rate constant makes a continuous transition from Arrhenius to anti-Arrhenius behavior through a narrow Arrhenius-like zone. This behavior is seen not only for the native state of the protein, but also for native-like states generated by adding subdenaturing amounts of guanidine deuterochloride (GdnDCl). Moderately destabilizing concentrations of the denaturant (1.5 M GdnDCl) completely removes the Arrhenius-like feature from the temperature window employed. The Arrhenius to anti-Arrhenius transition can be explained by the heat capacity model where temperature strengthens ground state interactions, perhaps hydrophobic in nature. The effect of the denaturant may appear to arise from direct protein-denaturant interactions that are structure-stabilizing under subdenaturing conditions. The temperature distribution of rate constants under different stability conditions also suggests that the prefactor in Arrhenius-like relations is temperature dependent. Although the use of the transition state theory (TST) offers several challenges associated with data interpretation, the present results and a consideration of others published earlier provide evidence for complexity of ring-flip dynamics in proteins.

The alkali molten globule state of horse ferricytochrome c: observation of cold denaturation.

Here, we present the basic structural properties and the thermodynamic description of a previously unknown alkali molten globule state of horse "ferricytochrome c". Both sodium and guanidinium cations stabilize the alkali-denatured state at pH 13, presumably by a charge screening mechanism. The Na(+)-stabilized conformation (B state) clearly meets with the molecular organizational definition of the generic molten globule state. The B state exhibits highly cooperative thermal unfolding transitions monitored by both near and far-UV CD. Analyses of these transitions show substantial heat capacity change, suggesting that the hydrophobic effect contributes considerably to its energetic stability. At low salt concentration where molten globules are less stable, the B state undergoes reversible cold denaturation.

Extensive misfolding in the refolding reaction of alkaline ferrocytochrome C.

This work describes an extensively misfolded kinetic intermediate in the folding of horse ferrocytochrome c. Under absolute native conditions, the alkali-unfolded protein liganded with carbon-monoxide exhibits misfolding. The misfolded product, apparently an off-pathway intermediate, requires large-scale unfolding in order to have a chance to fold correctly to the native state. The rate of unfolding of the misfolded intermediate limits the overall rate of protein folding. The high level of observed misfolding possibly results from a failure of the polypeptide chain to achieve by stochastic search the transition state relevant for successful folding. Such misfolding may be analogous to the failure of a sizable set of proteins in the intracellular milieu to fold to the functionally active native state.

The alkali molten globule state of ferrocytochrome c: extraordinary stability, persistent structure, and constrained overall dynamics.

This paper describes the structural and dynamic properties of a hitherto uncovered alkali molten globule (MG) state of horse "ferrocytochrome c" (ferrocyt c). Several experimental difficulties mainly because of heme autoxidation and extraordinary stability of ferrocyt c have been overcome by working with the carbonmonoxide-bound molecule under extremely basic condition (pH 13) in a strictly anaerobic atmosphere. Structural and molecular properties extracted from basic spectroscopic experiments suggest that cations drive the base-denatured CO-liganded protein to the MG state. The stability of this state is approximately 5.2 kcal mol(-)(1), and the guanidinium-induced unfolding transition is sharp (m(g) approximately 2.3 kcal mol(-)(1) M(-)(1)), suggesting contents of rigid tertiary structure. Strategic experiments involving the measurement of the CO association rate to the base-denatured protein and intrachain diffusion rates measured by laser photolysis of CO indicate a substantially restricted overall motion and stiffness of the polypeptide chain in the MG state. Possible placement of the state in the folding coordinate of ferrocyt c is discussed.

Protein folding in classical perspective: folding of horse cytochrome c.

Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), respectively. But the large disparity in thermodynamic stability is not strongly reflected in their refolding rates. Cross-pH studies, where GdnHCl-unfolded states of neutral- and alkaline ferrocyt c are allowed to refold to the same final pH and denaturant concentration, indicate that the refolding rates are largely independent of the stability, configuration, ionization, and solvation of the initial unfolded state. Also, burst relaxation signals in cross-pH refolding runs show the same quantitative dependence on GdnHCl, suggesting that the earliest relaxation or reconfiguration of the chains must be the same and is independent of the initial equilibrium unfolded state. Analyses along the classical line indicate an early transition state where much less than a third of the protein surface that is buried in the native state becomes buried. The barrier energy is of the order of 10 k(B)T. The results, apparently inconsistent with the predictions of the funnel model, afford a mechanistic description of folding in which the folding time of small single-domain proteins is set by the time needed for the denatured polypeptide to search-find a nativelike topology.