Shift In Equilibrium Position Research Articles

A continuous spectrophotometric assay for glutathione-independent glyoxalases

We developed a novel continuous assay to quantitatively characterize the catalytic activity of type III methylglyoxalases, a family of enzymes that detoxify methylglyoxal. This assay is based on spectrophotometric detection of hemithioacetal which forms in the reversible reaction of methylglyoxal with dithiothreitol. Due to rapid interconversion between hemithioacetal and methylglyoxal and the known equilibrium constant, hemithioacetal can be quantified spectrophotometrically at 286 nm and used as a reporter for methylglyoxal. When the concentration of methylglyoxal decreases due to catalytic conversion by methylglyoxalases, the concentration of hemithioacetal concomitantly decreases due to its spontaneous decomposition driven by the shift in equilibrium position. Therefore, the rate of total methylglyoxal consumption is the sum of the rate of hemithioacetal decomposition determined spectrophotometrically and the rate of change of methylglyoxal calculated from known concentrations of hemithioacetal. Varying concentrations of dithiothreitol and methylglyoxal creates a broad range of free methylglyoxal in solution that is crucial for the reliable determination of Michaelis constants. We demonstrate the utility of this assay using several recombinant glyoxalases for which kinetic parameters have been determined. This cost-effective and simple assay offers advantages over the existing discontinuous methods and will be useful for quantitative characterization of catalytic activities of type III methylglyoxalases.

The apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals

Loss-of-function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however, DJ-1 has been suggested to be a GSH-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is ∼500 m-1 at 37 °C and that the half-life of the resulting hemithioacetal is only 12 s. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Furthermore, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1-mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas-generated DJ-1-knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.

Bubble migration in two-dimensional foam sheared in a wide-gap Couette device: Effects of non-Newtonian rheology

We report experiments on the migration of a large bubble in an otherwise monodisperse two-dimensional (2D) foam sheared in a wide-gap Couette device. The bubble migrates away from the walls toward an equilibrium position between the center of the gap and the inner cylinder. This differs from the situation in a narrow-gap Couette device, where the equilibrium position is at the center of the gap [Mohammadigoushki and Feng, Phys. Rev. Lett. 109, 084502 (2012)]. The shift in equilibrium position is attributed to the non-Newtonian rheology of the foam, which is brought out by the nonhomogeneous shearing in a wide-gap geometry. Two aspects of the rheology, shear-thinning and the first normal stress difference, are examined separately by comparing with bubble migration in a xanthan gum solution and a Boger fluid. Shear-thinning shifts the equilibrium position inward while the normal stress does the opposite. Bubble migration in the 2D foam is the outcome of the competition between the two effects.

Quantitative Interpretation of Chemical Shifts Enables Mapping Proteins Conformational Landscape

To carry out their function, most proteins need to undergo transitions among different states. Understanding how the equilibrium between different states is altered upon ligand binding or interaction with other biological partners is of critical importance to explain protein function.Nuclear Magnetic Resonance isotropic 1H and 15N chemical shifts are a sensitive probe for both protein structure and conformational dynamics, and thus are an ideal observable to monitor equilibrium shifts. Yet, noise in the peak positions complicates the quantitative correlation of chemical shifts to the equilibrium position. Here we propose a robust methodology that relies on statistical analysis of NMR 1H/15N HSQC data and provides a quantitative measure of the equilibrium shift associated to ligand or substrate binding. In short, linearity in the 1H/15N chemical shifts position across different protein constructs is first identified by principal component analysis. Then, the scores of all residues on their principal components are filtered and combined together, and the shift in equilibrium position for each state is given in terms of confidence intervals. Finally, the degree of collectiveness of the protein response to ligand binding is mapped onto the protein structure by applying an adapted version of the chemical shifts covariance analysis (CHESCA). We illustrate the method in its applications to ligand binding to protein kinase A (PKA) and to a series of mutants of phospholamban. The results show that the method is capable of discerning the multiple states populated upon binding of different ligands to PKA as well as the shift in equilibrium position induced by mutations of phospholamban serine 16. Noticeably, the CHESCA analysis shows that while PKA response is highly collective throughout the whole protein, the PLN response is collective only in the surroundings of the perturbed region (domain 1A).

Passive elastic properties of the rat ankle

Passive properties of muscles and tendons, including their elasticity, have been suggested to influence motor control. We examine here the potential role of passive elastic muscle properties at the rat ankle joint, focusing on their potential to specify an equilibrium position of the ankle. We measured the position-dependent passive torques at the rat ankle before and after sequential cuts of flexor (a.k.a. dorsiflexor) and extensor (a.k.a. plantarflexor) ankle muscles. We found that there was a passive equilibrium position of the ankle that shifted systematically with the cuts, demonstrating that the passive torques produced by ankle flexor and extensor muscles work in opposition in order to maintain a stable equilibrium. The mean equilibrium position of the intact rat ankle ranged from 9.3° to 15.7° in extension relative to the orthogonal position, depending on the torque metric. The mean shift in equilibrium position due to severing extensors ranged from 4.4° to 7.7°, and the mean shift due to severing flexors was smaller, ranging from 0.9° to 2.5°. The restoring torques generated by passive elasticity are large enough (approximately 1.5–5mNm for displacements of 18° from equilibrium) to affect ankle movement during the swing phase of locomotion, and the asymmetry of larger extension vs. flexion torques is consistent with weight support, demonstrating the importance of accounting for passive muscle properties when considering the neural control of movement.

PKa‐Determination and Conformational Studies by NMR Spectroscopy of D‐Altrose‐Containing and other Pseudodisaccharides as Glycosidase Inhibitor Candidates

AbstractThe pKa values of six amine‐linked pseudodisaccharides have been determined by using an NMR‐based titration method. The pKa varies significantly depending on the linkage position and is inversely correlated with the number of electronegative groups in the β‐position to the amino group. Four of the pseudodisaccharides contain α‐altroside moieties, the conformations of which were determined in the protonated and neutral states, again by using NMR techniques. In the neutral state, 2‐amino‐2‐deoxyaltrosides and 3‐amino‐3‐deoxyaltrosides were both found to occupy 4C1 and OS2 conformations. On protonation, little change in the population distribution was seen for the 3‐amino‐3‐deoxyaltrosides, but for the 2‐amino‐2‐deoxyaltrosides, a shift in equilibrium position towards the skew conformer (more than 80 % populated) takes place, and also a small amount of the other chair conformer (i.e., 1C4, approximately 10 % populated) was observed. 3‐Amino‐3‐deoxyaltrosides have been shown to act as glycosidase inhibitors and insights into conformational equilibria as a function of protonation state should facilitate the design of better glycosidase inhibitors based on flexible monosaccharide amines.

The case for an internal dynamics model versus equilibrium point control in human movement.

The equilibrium point hypothesis (EPH) was conceived as a means whereby the central nervous system could control limb movements by a relatively simple shift in equilibrium position without the need to explicitly compensate for task dynamics. Many recent studies have questioned this view with results that suggest the formation of an internal dynamics model of the specific task. However, supporters of the EPH have argued that these results are not incompatible with the EPH and that there is no reason to abandon it. In this study, we have tested one of the fundamental predictions of the EPH, namely, equifinality. Subjects learned to perform goal-directed wrist flexion movements while a motor provided assistance in proportion to the instantaneous velocity. It was found that the subjects stopped short of the target on the trials where the magnitude of the assistance was randomly decreased, compared to the preceding control trials (P = 0.003), i.e. equifinality was not achieved. This is contrary to the EPH, which predicts that final position should not be affected by external loads that depend purely on velocity. However, such effects are entirely consistent with predictions based on the formation of an internal dynamics model.

Modeling of vibronic interaction and chaotic dynamics in electronic and nuclear subsystems of molecules

AbstractThe interaction of electronic and nuclear subsystems of a molecular system is described to be a consequence of cubic and biquadratic coupling on coordinates between electronic and nuclear oscillators in the classic and mixed quantum–classic models of a molecule. The simple relationships between two parameters of nonlinearity and usually used in the description of adiabatic vibronic interaction in a real molecules a shift of equilibrium position of nuclei and change of force constant them harmonic vibrations upon electronic excitation of a molecule are established. It is shown that performance of these relationships averaged on “fast” time dynamics of excitation of electronic oscillators of the classic model coincides with dynamics of population of the excited electronic state of the mixed quantum–classic model for any values of nuclear positions. The influence considered vibronic interaction on the character of dependence of quasienergies of a molecule dressed by a cloud of photons from the coordinate nuclear oscillator is investigated. As a necessary condition for the appearance of chaotic dynamics of nuclei in the considered quantum–classic model of a molecule at transformation by its light fields it is proposed to consider the occurrence of two‐well quasienergy. Taking account this criterion, it is shown that only the considered cubic (but not biquadratic) vibronic interaction is one of the reasons for the chaotic dynamics of nuclei in molecules interacting with a light field. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002

Observation of single transits in supercooled monatomic liquids.

A transit is the motion of a system from one many-particle potential energy valley to another. We report the observation of transits in molecular dynamics calculations of supercooled liquid argon and sodium. Each transit is a correlated simultaneous shift in the equilibrium positions of a small local group of particles, as revealed in the fluctuating graphs of the particle coordinates versus time. To the best of our knowledge, this is the first reported direct observation of transit motion in a monatomic liquid in thermal equilibrium. We found transits involving 2-11 particles, having mean shift in equilibrium position on the order of 0.4R(1) in argon and 0.25R(1) in sodium, where R1 is the nearest neighbor distance. The time it takes for a transit to occur is approximately one mean vibrational period, confirming that transits are fast.

Intraligand Charge Transfer in Pt(qol)(2). Characterization of Electronic States by High-Resolution Shpol'skii Spectroscopy.

Pt(qol)(2) (qol(-) = 8-quinolinolato-O,N) is investigated in the Shpol'skii matrices n-heptane, n-octane-h(18), n-octane-d(18), n-nonane, and n-decane, respectively. For the first time, highly resolved triplet phosphorescence as well as triplet and singlet excitation spectra are obtained at T = 1.2 K by site-selective spectroscopy. This permits the detailed characterization of the low-lying singlet and triplet states which are assigned to result mainly from intraligand charge transfer (ILCT) transitions. The electronic origin corresponding to the (3)ILCT lies at 15 426 cm(-)(1) (FWHM approximately 3 cm(-)(1)) exhibiting a zero-field splitting smaller than 1 cm(-)(1), which shows that the metal d-orbital contribution to the (3)ILCT is small. At T = 1.2 K, the three triplet sublevels emit independently due to slow spin-lattice relaxation (slr) processes. Therefore, the phosphorescence decays triexponentially with components of 4.5, 13, and 60 &mgr;s. Interestingly, two of the sublevels can be excited selectively, which leads to a distinct spin polarization manifested by a biexponential decay. At T = 20 K, the decay becomes monoexponential with tau = 10 &mgr;s due to a fast slr between the triplet sublevels. From the Zeeman splitting of the (3)ILCT the g-factor is determined to be 2.0 as expected for a relatively pure spin triplet. The (1)ILCT has its electronic origin at 18 767 cm(-)(1) and exhibits a homogeneous line width of about 12 cm(-)(1). This feature allows us to estimate a singlet-triplet intersystem crossing rate of about 2 x 10(12) s(-)(1). This relatively large rate compared to values found for closed shell metal M(qol)(n)() compounds displays the importance of spin-orbit coupling induced by the heavy metal ion. Moreover, this small admixture leads to the relatively short emission decay times. All spectra show highly resolved vibrational satellite structures. These patterns provide information about vibrational energies (which are in good accordance with Raman data) and shifts of equilibrium positions between ground and excited states. These shifts are different in the (1)ILCT and (3)ILCT states. The vibrational satellite structures support the assignment of ILCT character to the lowest excited states.

Rate processes on moderately smooth surfaces

To describe the effect of lateral interactions between adsorbed particles on the kinetics of rate processes on uniform surfaces, the lattice–gas models are usually employed. Traditionally, these models are based on the idea that the equilibrium positions of adsorbed particles are fixed rigidly on the surface. This assumption may be incorrect in many real systems (including such traditional systems as CO or hydrogen on different metals), where the activation energy for surface diffusion is not very high (or is low) compared to lateral interactions between nearest-neighbor adsorbed particles (i.e., where the surface is smooth). In the present paper, a general theory is developed to take into account the influence of shifts of equilibrium position of adparticles on the kinetics of various rate processes on solid surfaces. The theory is applicable to the case when the activation energy for surface diffusion exceeds lateral interactions between adparticles. The shifts of equilibrium positions are shown to result in significant changes in the rate constants of various processes. The effect of shifts on the coverage dependence of the apparent Arrhenius parameters for desorption, thermal desorption spectra, and the coverage dependence of the chemical diffusion coefficient is also demonstrated in detail.