Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonas aeruginosa

The irreversible thermal denaturation of cytochrome cd₁ oxidase from $\text{P.}\text{aeruginosa}$ as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10^?3s^?1; oxidized, 3 × 10^?3s^?1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).     

Repurposing of FDA-approved drugs to target MurB and MurE enzymes in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is one amongst the top 10 causes of death worldwide. The growing rise in antibiotic resistance compounded with slow and expensive drug discovery has further aggravated the situation. ‘Drug repurposing’ is a promising approach where known drugs are examined for a new indication. In the present study, we have attempted to identify drugs that could target MurB and MurE enzymes involved in the muramic acid synthesis pathway (Mur Pathway) in Mtb. FDA-approved drugs from two repositories i.e. Drug Bank (1932 drugs) and e-LEA3D (1852 drugs) were screened against these proteins. Several criteria were applied to study the protein-drug interactions and the consensus drugs were further studied by molecular dynamics (MD) simulation. Our study found Sulfadoxine (–7.3?kcal/mol) and Pyrimethamine (–7.8?kcal/mol) to show stable interaction with MurB while Lifitegrast (–10.5?kcal/mol) and Sildenafil (–9.1?kcal/mol) showed most reliable interaction with MurE. Furthermore, binding free energy (ΔG_bind), RMSD and RMSF data and the number of hydrogen bonds corroborated the stability of interactions and hence these drugs for repurposing should be explored further.

Communicated by Ramaswamy H. Sarma     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Influence of hydrogen bonds on edge-to-face interactions between pyridine molecules

Edge-to-face interactions between two pyridine molecules and the influence of simultaneous hydrogen bonding of one or both of the pyridines to water on those interactions were studied by analyzing data from ab initio calculations. The results show that the edge-to-face interactions of pyridine dimers that are hydrogen bonded to water are generally stronger than those of non-H-bonded pyridine dimers, especially when the donor pyridine forms a hydrogen bond. The binding energy of the most stable edge-to-face interacting H-bonded pyridine dimer is ?5.05 kcal/mol, while that for the most stable edge-to-face interacting non-H-bonded pyridine dimer is ?3.64 kcal/mol. The interaction energy data obtained in this study cannot be explained solely by the differences in electrostatic potential between pyridine and the pyridine–water dimer. However, the calculated cooperative effect can be predicted using electrostatic potential maps.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Isolation and characterization of a molybdenum iron-sulfur protein from Desulfovibrio africanus.

A molybdenum-containing iron-sulfur protein has been isolated from the sulfate reducer $\text{Desulfovibrio}\text{africanus}$. The protein appears to be a complex protein of high molecular weight (112,000) composed of 10 subunits (mol. wt. 11,500) and containing a high amount of molybdenum (5–6 atoms/mole) with approx. 20 atoms each of iron and labile sulfide. The spectrum shows peaks at around 615, 410 and 325 nm with a protein peak at 280 nm. Its millimolar extinction coefficients at 615, 410 and 280 nm are 48.4, 64.4 and 141 respectively. The protein contains 106 amino-acid residues per subunit of mol. wt. 11,262 and the number of cysteine residues is 2 per subunit. The N-terminal sequence which has been determined up to 26 residues is characterized by its high degree of hydrophobicity.     

Spin labels as enzyme substrates Heterogeneous lipid distribution in liver microsomal membranes

Phenobarbital-stimulated microsomal membranes of rabbit liver, containing the cytochrome P450- cytochrome P450 reductase hydroxylating enzyme system in high concentration, have been studied with a version of the spin label technique which uses nitroxide radicals as enzyme substrates. The reduction kinetics of a phosphate ester of tetramethylpiperidine nitroxide (TEMPO-phosphate) and of stearic acid nitroxide by the cytochrome P450 reductase has been studied as a function of the temperature. The Arrhenius plot of the reduction rate constants reveals a striking difference in the behaviour of the water-soluble TEMPO-phosphate label and the lipid-soluble fatty acid label: The activation energy of the fatty acid reduction decreases abruptly at about 32°C from a value of 30.8 kcal/mole to a value of 8.7 kcal/mole, whereas no such break is observed in the Arrhenius plot of the TEMPO-phosphate reduction which yields a value of the activation energy of $\text{ΔW = 13.8}$ kcal/mole in the whole temperature range investigated. Our results clearly indicate the existence of a mosaic-like structure of the membrane with the whole enzyme system being enclosed by a rather rigid phospholipid halo which is in a quasicrystalline structure below 32 °C and undergoes a crystalline-liquid crystalline phase transition at 32 °C, while the bulk lipid of the membrane is in a rather fluid state as reflected by the measured high diffusion coefficient of $\text{D}{}_{\mathrm{<mtext>diff</mtext>}}\text{= 11.0·10}{}^{\mathrm{?8}}\text{cm}{}^2\text{/s}$ at 30 °C and low activation energy of diffusion of $\text{ΔW = 3.85}\text{kcal/mole}$ of a fatty acid spin label incorporated in the membrane.     

Application of ab initio molecular-orbital calculations to the structural moieties of carbohydrates. Part VI

Ab initio RHF/4–31G molecular-orbital calculations have been conducted on methoxymethyl formate and methoxymethyl acetate as models for examining the anomeric effect and stereochemistry of 1-O-acetylglycopyranoses. The results indicate that, as with the methyl glycopyranosides, the α-⁴C₁(D) configurations are more stable than the β-⁴C₁(D), except that the energy difference is more dependent on the disposition about the glycosidic bond. The lowest-energy conformations occur with glycosidic torsion-angles of ?  180°, where the anomeric energy is about 4 kcal/mol. There is a secondary energy-minimum at ?  90°, for which the anomeric energy is less, about 2 kcal/mol. This orientation corresponds to the conformation most commonly observed in the crystal structures of peracetylated glycopyranoses. Small differences in the CO single-bond lengths, which are observed experimentally in both the α and β anomers, are reproduced by the theoretical calculations.     

Tetanus relaxation. Temperature effects and Arrhenius analysis

Time constants (τ) have been accurately measured for the exponentially falling, latter 60–70% of the relaxation phase of maximal isometric tetani of the mouse extensor digitorum longus muscle over the range 15–35°C. Corresponding to the τ values, the rate constants (k = 25.0?189 s^-1) are assumed to describe a temperature-sensitive, first-order, rate-limiting reaction underlying, and determining the kinetics of, muscle relaxation. The mean Arrhenius plot for the k values of 6 muscles consists of 2 linear segments with a 25°C transition temperature. The activation energies at the relatively lower and higher temperatures are 22.9 and 12.6 kcal/mol, respectively. These values are qualitatively, and to some extent quantitatively, similar to corresponding known Arrhenius results of the Ca²⁺ active transport mechanism and the physical properties of the membrane of isolated sarcoplasmic reticulum. Thus, the present findings strongly indicate that relaxation of living muscle critically involves the ‘relaxing factor’ activity of Ca²⁺ uptake, as previously inferred from research on isolated sarcoplasmic reticulum. Using transition-state theory, the Arrhenius results indicate that $\text{ΔG}{}^3$ of the assumed rate-limiting reaction is 14.8–15.0 kcal/mol at all temperatures studied, and $\text{ΔS}{}^3$ is about 25 and ?10 cal/degree per mol at temperatures below and above the transition temperature, respectively. These also correspond, at least qualitatively, to the values of the activation thermodynamic parameters of isolated sarcoplasmic reticulum. The negative $\text{ΔS}{}^3$ at the higher temperature range, denoting an increase in order associated with the assumed activation process of the Ca²⁺ transport system, requires clarification.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Coordination of adenylate energy charge and phosphorylation state during ischemia and under physiological conditions in rat liver and kidney

The relation of the adenylate energy charge $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP/ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ to the phosphorylation state (ATP)/(ADP)(HPO₄^2?) in rat liver and kidney was analyzed. Under physiological conditions and in ischemia, the two regulatory parameters, calculated from reported values for adenine nucleotides and inorganic phosphate (P_i) and from new observations, were closely coordinated. Energy charge was an inverse linear function of P_i and -log (1 - energy charge) was a positive linear function of log phosphorylation state. To evaluate experimental data with known energy charge, but unknown P_i, and to determine the theoretical relation between energy charge and phosphorylation state, P_i was estimated from a) the regression equation: P_i, μmol/g wet wt tissue = 1.05 - energy charge/0.073 and b) the empirical relationship: (P_i/2P_a) + energy charge = k, where P_a = σAMP + 2ADP + 3ATP and k = 1. With both estimates, the relation between phosphorylation state and energy charge for the experimental data was, within error, the same as that observed with measured P_i and concordant with theoretical values. Over the physiological range of energy charge (～0.85 – 0.95, log phosphorylation state ～3.3 – 4.3), apparent ΔG_ATP (×2) was closer to the range of ΔG observed by Wilson $\text{et al}$ (Biochem. J. $\text{140}$:57, 1974) for transfer of two electrons from mitochondrial NAD to the cytochrome c couple than the ΔG_ATP (×2) they reported, supporting their conclusion that near-equilibrium exists between the mitochondrial respiratory chain and the cytoplasmic phosphorylation state under physiological conditions. From evidence presented, it is postulated that the phosphorylation state is regulated by the adenylate energy charge.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Degradation of surface-labeled hepatoma membrane polypeptides: Effect of inhibitors

When their membrane proteins were labeled with ¹²⁵I by lactoperoxidase, dividing hepatomacells lost radio activity to the medium in a biphasic manner ($\text{T}\text{1}\text{2}\text{= 16–26}\text{h}\text{, > 40}\text{h}$). Lysosomotropic weak bases, chloroquine, and NH₄Cl inhibited the rapid phase by 59%. More than 50% of the radio activity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60–71% inhibited by lysosomotropic agents chloro quine and NH₄Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH₄Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-l-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by21–24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 °C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lyososomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to, agents which disrupt the cytoskeleton.     

Ab Initio Quantum Mechanics Analysis of Imidazole C-H…O Water Hydrogen Bonding and a Molecular Mechanics Forcefield Correction

Abstract

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but ‘activated’ C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H…O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H…0 and C—O. In general, these calculations indicate that C-H…0 interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) level of theory of a water C-H…O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H…O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole C_e?H…O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole C_e?H…O water and imidazole N_d?H…O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H…O water interaction, these forcefields do not adequately account for the imidazole C_e?H…O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H…O hydrogen bonded complex.     

Computational study on C−H…π interactions of acetylene with benzene, 1,3,5-trifluorobenzene and coronene

Meta-hybrid density functional theory calculations using M06-2X/6-31+G(d,p) and M06-2X/6-311+G(d,p) levels of theory have been performed to understand the strength of C?H^…π interactions of two possible types for benzene-acetylene, 1,3,5-trifluorobenzene-acetylene and coronene-acetylene complexes. Our study reveals that the C?H^...π interaction complex where acetylene located above to the center of benzene ring (classical T-shaped) is the lowest energy structure. This structure is twice more stable than the configuration characterized by H atom of benzene interacting with the π-cloud of acetylene. The binding energy of 2.91 kcal/mol calculated at the M06-2X/6-311+G(d,p) level for the lowest energy configuration (1A) is in very good agreement with the experimental binding energy of 2.7?±?0.2 kcal/mol for benzene-acetylene complex. Interestingly, the C?H^...π interaction of acetylene above to the center of the aromatic ring is not the lowest energy configuration for 1,3,5-trifluorobenzene-acetylene and coronene-acetylene complexes. The lowest energy configuration (2A) for the former complex possesses both C?H^...π interaction and C?H^...F hydrogen bond, while the lowest energy structure for the coronene-acetylene complex involves both π-π and C?H^...π interactions. C?H stretching vibrational frequencies and the frequency shifts are reported and analyzed for all of the configurations. We observed red-shift of the vibrational frequency for the stretching mode of the C-H bond that interacts with the π-cloud. Acetylene in the lowest-energy structures of the complexes exhibits significant red-shift of the C?H stretching frequency and change in intensity of the corresponding vibrational frequency, compared to bare acetylene. We have examined the molecular electrostatic potential on the surfaces of benzene, 1,3,5-trifluorobenzene, coronene and acetylene to explain the binding strengths of various complexes studied here.     

Interrelations between pH and temperature for the catalytic rate of the M4 lsozyme of lactate dehydrogenase (EC 1.1.1.27) from goldfish (Carassius auratus L.).

A kinetic study of the rate of pyruvate reduction by goldfish LDH-M₄ (the homotetrameric form of lactate dehydrogenase which predominates in skeletal muscle) provided an analysis of the effects of pH and temperature on v (reaction velocity) and estimates of how temperature might affect catalysis in vivo, where the physiological pH regulation imposes a temperature coefficient of ?0.015 to ?0.020 pH unit/ °C. Consistent with published data for other LDHs, (i) V (maximum reaction velocity) was pH insensitive over a physiological pH range, (ii) the K_m for pyruvate, K_P, was sensitive to both pH and temperature, and (iii) the K_m for NADH and the dissociation constant for NADH were both sensitive to temperature, but not to pH. V approximately doubled with each 10 °C (E_a = 11.7 kcal/mol). The effects of pH and temperature on K_P were consistent with two enthalpic contributions, an ionization enthalpy (ΔH_i^°) of 7.2 kcal/mol (probably a histidine imidazole), and an enthalpy (ΔH_SO) of 5.8 kcal/mol for the combination of pyruvate with the nonionized (pH ? pK′) LDH-NADH complex. When the pH was varied according to the physiological temperature coefficient, v was more sensitive to temperature than for conditions of constant pH, the usual design of kinetic experiments. This effect was due to the decreased temperature sensitivity of K_P caused by partial concellation of the ΔH_i^° effect by the pH regulation: $\text{d}\text{pH}\text{dT}\text{?}\text{d}\text{p}\text{K′}\text{dT}$. At constant pH, on the other hand, K_P increased strongly with temperature and had the effect of offsetting (especially at higher pH values) the large increases in V. It was suggested that the magnitudes of ΔH_i^° and ΔH_SO might have been important in the evolution of LDHs and other enzymes of cold-blooded animals.     

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, Δ$\text{G}$°, at 10 and 100 mM ionic strength (?7.85 and ?8.26 kcal/mol, respectively). Binding saturates at an area of 650 Ų per melittin molecule. A change in the surface dipole potential corresponding to ?1.1 debye/?_a ($\text{?}{}_{\mathrm{a}}\text{= dielectric constant of the adsorption region}$) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.     

Difference in microviscosity induced by different cholesterol levels in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells

Microviscosity ($\text{}\text{?}$gh) in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells, and in liposomes prepared from their lipid extracts, was determined with the aid of the fluorescence polarization properties of 1,6-diphenyl 1,3,5-hextriene embedded in it. The $\text{}\text{?}$gh values, both in intact cells and in the liposomes, are distinctively greater for normal lymphocytes than for the lymphoma cells, whereas the fusion activation energy in both types of cells and liposomes is 8 ± 0.5 kcal/mol. Determination of cholesterol revealed that its relative amount in a lymphoma cell is about half of that of a normal lymphocyte, a difference that may account for the above difference in fluidity. This thesis is supported by the observed changes in $\text{}\text{?}$gh, which follow artificial changes in cholesterol contents in the surface membrane of both cell types. Introduction of exogeneous cholesterol into the cell surface membranes was performed with lecithin-cholesterol (1:1) liposomes, and in lymphoma cells resulted in an increase of $\text{}\text{?}$gh to a level of normal lymphocytes. Extraction of native cholesterol from the cell surface membranes was carried out with lecithin liposomes, and in normal lymphocytes results in a decrease of $\text{}\text{?}$gh to a value similar to that of lymphoma cells. The induced changes in cholesterol contents are practically reversible for both cell types. By virtue of controlling the microviscosity of lipid layers, the level of cholesterol in cell surface membranes may play an important role in determining biological activities of normal and malignant cells.     

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively filled by a reversible consecutive dissociation-unfolding mechanism according to Na = 4M ? 4M* (where N is the native letramer, and M and M* two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101). At P ? 1 kbar, the pressure deactivalion of both isoenzymes (H₄ and M₄) is described by the two-state equilibrium N ? 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M₄: Δg_Diss = 110 $\text{kJ}\text{mol}$, ΔSDiss = ?860 J/K per mol, ΔH_Diss = ?124 $\text{kJ}\text{mol}$ (enzyme concentration 10 $\text{μg}\text{ml}$, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be ΔV_Diss = ?420 $\text{ml}\text{mol}$ (0.7 < p < 0.9 kbar). Measurements using 8-anilino-1-naphlhalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurizarion beyond 1 kbar. Within the range of reversible deactivation (p < 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. ²H₂O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.