Deuterium kinetic isotope effects on the dissociation of a protein-fatty acid complex in the gas phase

Deuterium kinetic isotope effects (KIEs) are reported for the first time for the dissociation of a protein-ligand complex in the gas phase. Temperature-dependent rate constants were measured for the loss of neutral ligand from the deprotonated ions of the 1:1 complex of bovine β-lactoglobulin (Lg) and palmitic acid (PA), (Lg + PA)(n-) → Lg(n-) + PA, at the 6- and 7- charge states. At 25 °C, partial or complete deuteration of the acyl chain of PA results in a measurable inverse KIE for both charge states. The magnitude of the KIEs is temperature dependent, and Arrhenius analysis of the rate constants reveals that deuteration of PA results in a decrease in activation energy. In contrast, there is no measurable deuterium KIE for the dissociation of the (Lg + PA) complex in aqueous solution at pH 8. Deuterium KIEs were calculated using conventional transition-state theory with an assumption of a late dissociative transition state (TS), in which the ligand is free of the binding pocket. The vibrational frequencies of deuterated and non-deuterated PA in the gas phase and in various solvents (n-hexane, 1-chlorohexane, acetone, and water) were established computationally. The KIEs calculated from the corresponding differences in zero-point energies account qualitatively for the observation of an inverse KIE but do not account for the magnitude of the KIEs nor their temperature dependence. It is proposed that the dissociation of the (Lg + PA) complex in aqueous solution also proceeds through a late TS in which the acyl chain is extensively hydrated such that there is no significant differential change in the vibrational frequencies along the reaction coordinate and, consequently, no significant KIE.     

Deuterium isotope effects in solutions of 2,4-pentanedione

Proton and deuterium n.m.r. measurements in acetylacetone and 3,3-d₂-acetylacetone are reported. Deuterium-isotope effects on chemical shifts and keto–enol equilibria as a function of concentration in the two solvents triethylamine and pyrrole are determined. A strong solvent concentration dependence for d-isotope effects is observed in triethylamine; in pyrrole no dependence is obtained. The results are interpreted in terms of an equilibrium between the symmetrical (C_2v) and asymmetrical (C_s) enolic forms in acetylacetone. The usefulness of ²H n.m.r. for the study of exchange processes is demonstrated.     

Deuterium isotope effects on 13C chemical shifts of nitromalonamide

The OH chemical shift of the enol form of nitromalonamide is found at 18.9 ppm both in DMSO-d(6) and in DMF-d(7) indicating a very strong hydrogen bond. The OH chemical shift is insensitive to temperature changes. Contrary to the large OH chemical shift, a small two-bond deuterium isotope effect of 0.135 ppm due to deuteration at the OH position is found at the enolic carbon. This is confirmed by density functional theory calculations. The observed effects are interpreted as due to an equilibrium between identical enolic forms. These show a strong OH...O hydrogen bond as well as a NH...O-N=O hydrogen bond.     

Deuterium isotope effects in liquid–liquid phase diagrams of aniline + cyclohexane mixtures

Liquid–liquid miscibility temperatures as a function of composition and deuterium substitution have been experimentally determined for binary mixtures of aniline and cyclohexane and their various deuterated forms: aniline-d₂, aniline-d₅, cyclohexane-d₁₂. In all cases studied, the deuteration leads to the upward shift of the upper critical solution temperature. This effect is discussed in the frame of the theory of the condensed phase isotope effects coupled with the phenomenological g^E thermodynamic model of solutions.     

Deuterium isotope effects on one-bond carbon,hydrogen coupling constants

Small, negative deuterium isotope effects on ¹J(CH) have been found for a variety of simple organic molecules. These new experimental data support theoretical calculations and suggestions based on previous imprecise experimental measurements. The deuterium isotope effects upon the ¹³C chemical shifts were also measured with high precision.     

Enthalpy-entropy compensation in the effects of urea on hydrophobic interactions

By comparison of neopentane pair potentials of mean force (PMFs) in room temperature water and 6.9 molar aqueous urea, it was recently shown that urea molecules affect the PMF minima in an unexpected way (Lee, M.-E.; van der Vegt, N. F. A. J. Am. Chem. Soc. 2006, 128, 4948). While the first PMF minimum in urea solution has an identical shape and depth to those of the corresponding minimum in water, the second minimum in urea solution is broader, deeper, and shifted out to a slightly larger distance. Here, we present a study of the enthalpic and entropic contributions to these PMFs. Its significance for understanding the driving forces responsible for thermodynamically favorable neopentane contact and solvent-separated distances in urea solution is discussed. We propose that the solute-solvent entropy and solute-solvent enthalpy changes should be analyzed for obtaining an unambiguous molecular-scale picture. In urea solution, enthalpy-entropy compensation effects associated with structural solvent reorganization processes are large, causing changes of the system's enthalpy and entropy with hydrophobic pair separation to be very different from the solute-solvent enthalpy and entropy changes. The entropies are discussed in terms of the molecular-scale solvent reorganization processes.     

Deuterium kinetic isotope effects in microsolvated gas-phase E2 reactions

This work describes the first experimental studies of deuterium kinetic isotope effects (KIEs) for the gas-phase E2 reactions of microsolvated systems. The reactions of F(-)(H(2)O)(n) and OH(-)(H(2)O)(n), where n = 0, 1, with (CH(3))(3)CX (X = Cl, Br), as well as the deuterated analogs of the ionic and neutral reactants, were studied utilizing the flowing afterglow-selected ion flow tube technique. The E2 reactivity is found to decrease with solvation. Small, normal kinetic isotope effects are observed for the deuteration of the alkyl halide, while moderately inverse kinetic isotope effects are observed for the deuteration of the solvent. Minimal clustering of the product ions is observed, but there are intriguing differences in the nature and extent of the clustering process. Electronic structure calculations of the transition states provide qualitative insight into these microsolvated E2 reactions.     

Deuterium isotope effect on charge-transfer fluorescence

Quantum yields and decay times of fluorescence of charge-transfer complexes of tetracyanoethylene (an electron acceptor) with protonated and deuterated aromatic hydrocarbon donors were measured. The deuterium isotope effect on radiationless transition (identified as the

internal conversion) was observed. This observation is taken as evidence of the dominant role of intramolecular within the donor and/or the acceptor molecule) vibrations in radiationless transitions from excited charge-transfer states of molecular complexes.     

Critical importance of length-scale dependence in implicit modeling of hydrophobic interactions

The existence of length-scale dependence of hydrophobic solvation has important implications in the equilibrium of disordered, partially folded, and folded protein conformations. Neglecting this dependence, such as in popular solute surface-area based implicit solvent models with fixed surface tension coefficients, severely limits the ability to accurately model protein conformational equilibrium. We illustrate such fundamental limitations by examining the potentials of mean force of forming dimeric and trimeric nonpolar clusters and propose a new empirical model that effectively captures the context dependence of the local effective surface tension. Further optimization of the new model with other components of the implicit solvent force fields provides promise to significantly improve one's ability to simulate protein folding and conformational transitions. The existence of length-scale dependence of hydrophobic solvation has important implications in the equilibrium of disordered, partially folded, and folded protein conformations. Neglecting this dependence, such as in popular solute surface-area based implicit solvent models with fixed surface tension coefficients, severely limits the ability to accurately model protein conformational equilibrium. We illustrate such fundamental limitations by examining the potentials of mean force of forming dimeric and trimeric nonpolar clusters and propose a new empirical model that effectively captures the context dependence of the local effective surface tension. Further optimization of the new model with other components of the implicit solvent force fields provides promise to significantly improve one's ability to simulate protein folding and conformational transitions.     

Deuterium isotope effects on the interaction between hyperbranched polyethylene imine and an anionic surfactant

Solvent isotope effects on the interaction between the hyperbranched cationic polyelectrolyte, polyethylene imine (PEI), and the anionic surfactant sodium dodecyl sulfate (SDS) were investigated using potentiometric titration and eletrophoretic mobility measurements. In the basic pH range, a significantly higher fraction of the amine groups was found to be protonated when the PEI was dissolved in D2O compared to H2O at the same pH/pD. The difference in polymer charge in the two solvents decreases gradually with decreasing pH, and it completely diminishes at around pH = 4. Electrophoretic mobility measurements of PEI/SDS complexes at different pH values correlated very well with these observations. At pH/pD approximately 9 a much higher mobility of the PEI/SDS complexes was found in D2O than in H2O at low surfactant concentrations, and the charge neutralization point shifted to a considerably larger surfactant concentration in heavy water. These results can be explained by the significantly higher charge density of the PEI in D2O compared to H2O. However, at the natural pH/pD as well as at pH = 4 and pD = 4 conditions the PEI molecules have roughly equal charge densities, which result in very similar charged characteristics (mobilities) of the PEI/SDS complexes as well as the same charge neutralization SDS concentration. It can be concluded that extreme care must be taken in the general analysis of those experiments in which weak polyelectrolyte/surfactant aggregates are investigated in heavy water, and then these observations are correlated with structures of the same system in water.     

Deuterium isotope effects in the meta photocycloaddition of aromatic compounds to alkenes

Secondary deuterium isotope effects were observed in the meta photocycloaddition of several aromatic compounds to cyclopentene.     

Deuterium isotope effects on 13C and 15N nuclear shielding in o-hydroxyazo dyes

Deuterium isotope effects on the ¹³C and ¹⁵N nuclear shieldings of o-hydroxyazo compounds are described. Both the direct and the equilibrium contributions were determined. Large direct deuterium isotope effects on ¹⁵N nuclear shieldings for ¹Δ, ²Δ and ⁴Δ and negative Δ¹J(¹⁵NH) [D] values were observed. Isotope effects on ¹⁵N nuclear shieldings, because of their magnitudes, are shown to be very useful in determining changes in the azo–hydrazone equilibrium. Isotope effects on ¹³C nuclear shieldings are smaller, but the effects observed at the carbon resonances of the N-phenyl ring are likewise very useful in determining the shift in the equilibrium. Deuterium substitution leads in all cases to a shift in the equilibrium so that the content of the predominant form of the protio compound is increased.     

Deuterium isotope effects on 13C nuclear shielding as a measure of tautomeric equilibria

Large deuterium isotope effects of both signs have been observed on the ¹³C nuclear shielding in proton-chelating tautomeric forms of β-thioxoketones, and small isotope effects of both signs have been found in related forms of Schiff's bases of salicylaldehydes. These effects are interpreted in terms of shifts in the tautomeric equilibria on deuterium substitution of the enolic proton. The observation of deuterium isotope effects is suggested as a useful method for the detection of fast tautomeric equilibria in systems of the following type. .     

Deuterium isotope effects in 13C NMR spectroscopy: Stereochemical assignment of monodeuteriated allylic alcohols.

Vicinal deuterium isotope effects on 13C NMR chemical shifts, ³ΔC (Φ) are given for ten monodeuteriated allylic alcohols. The relationship found for nine of them: 3ΔC(0°) ≅ 5/3 x ³ΔC(180°) allows the stereochemical assignement of their deuterium labeling. This assignment is in agreement with that from the Karplus-type relationship of vicinal coupling constants, ³J_C-D(Φ).     

Molecular dynamics simulations of pressure effects on hydrophobic interactions.

We report results on the pressure effects on hydrophobic interactions obtained from molecular dynamics simulations of aqueous solutions of methanes in water. A wide range of pressures that is relevant to pressure denaturation of proteins is investigated. The characteristic features of water-mediated interactions between hydrophobic solutes are found to be pressure-dependent. In particular, with increasing pressure we find that (1) the solvent-separated configurations in the solute-solute potential of mean force (PMF) are stabilized with respect to the contact configurations; (2) the desolvation barrier increases monotonically with respect to both contact and solvent-separated configurations; (3) the locations of the minima and the barrier move toward shorter separations; and (4) pressure effects are considerably amplified for larger hydrophobic solutes. Together, these observations lend strong support to the picture of the pressure denaturation process proposed previously by Hummer et al. (Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1552): with increasing pressure, the transfer of water into protein interior becomes key to the pressure denaturation process, leading to the dissociation of close hydrophobic contacts and subsequent swelling of the hydrophobic protein interior through insertions of water molecules. The pressure dependence of the PMF between larger hydrophobic solutes shows that pressure effects on the interaction between hydrophobic amino acids may be considerably amplified compared to those on the methane-methane PMF.     

Deuterium kinetic isotope effects in microsolvated gas-phase E2 reactions: Methanol and ethanol as solvents

The gas-phase reactions of F(-)(CH(3)OH) and F(-)(C(2)H(5)OH) with t-butyl bromide have been investigated to explore the effect of the solvent on the E2 transition state. Kinetic isotope effects (KIEs) were measured using a flowing afterglow-selected ion flow tube (FA-SIFT) mass spectrometer upon deuteration of both the alkyl halide and the alcohol. Kinetic isotope effects are significantly more pronounced than those previously observed for similar reactions of F(-)(H(2)O) with t-butyl halides. KIEs for the reaction of F(-)(CH(3)OH) with t-butyl bromide are 2.10 upon deuteration of the neutral reagent and 0.74 upon deuteration of the solvent. KIEs for the reaction of F(-)(C(2)H(5)OH) with t-butyl bromide are 3.84 upon deuteration of the neutral reagent and 0.66 upon deuteration of the solvent. The magnitude of these effects is discussed in terms of transition-state looseness. Additionally, deuteration of the neutral regent and deuteration of the solvent do not produce completely separable isotope effects, which is likely due to a crowded transition state. These results are compared to our previous work on S(N)2 and E2 solvated systems.