Conformational Equilibrium Constants Research Articles

Quantifying Through‐Space Substituent Effects

AbstractThe description of substituents as electron donating or withdrawing leads to a perceived dominance of through‐bond influences. The situation is compounded by the challenge of separating through‐bond and through‐space contributions. Here, we probe the experimental significance of through‐space substituent effects in molecular interactions and reaction kinetics. Conformational equilibrium constants were transposed onto the Hammett substituent constant scale revealing dominant through‐space substituent effects that cannot be described in classic terms. For example, NO2 groups positioned over a biaryl bond exhibited similar influences as resonant electron donors. Meanwhile, the electro‐enhancing influence of OMe/OH groups could be switched off or inverted by conformational twisting. 267 conformational equilibrium constants measured across eleven solvents were found to be better predictors of reaction kinetics than calculated electrostatic potentials, suggesting utility in other contexts and for benchmarking theoretical solvation models.

An Organic Receptor Isolated in an Unusual Intermediate Conformation: Computation, Crystallography, and Hirshfeld Surface Analysis.

1,1″-1,4-Phenylene-bis(methylene)bis-4,4'-bipyridinium cation [C28H24N4]2+ (c), an organic receptor that generally crystallizes in its anti conformation, has recently been shown to be isolated in its syn conformation in an ion-paired compound [C28H24N4][Zn(dmit)2]·2DMF (dmit2- = 1,3-dithiole-2-thione-4,5-dithiolate; DMF = dimethylformamide). In this article, we demonstrated that the same receptor [C28H24N4]2+ (c) can also be stabilized in an unusual intermediate conformation (neither syn nor anti) with PF6- anion in compound [C28H24N4](PF6)2·(1,4-dioxane) (1·(1,4-dioxane)). The energetically favored anti conformation has been described in its nitrate salt [C28H24N4](NO3)2·2H2O (2·2H2O). Compounds 1·(1,4-dioxane) and 2·2H2O, crystallizing in triclinic and monoclinic systems with space groups P1̅ and P21/n, respectively, were additionally characterized by Hirshfeld surface analysis. The density functional theory calculations are performed to understand the internal mechanism of the stability of various conformers of cationic receptor c, compound 1, and compound 2. In conjunction with the electronic stability of the conformers, the natural bond orbital analysis and conformational equilibrium constants at different temperatures are also calculated to find out the sources of the different stability of the various conformers of experimentally synthesized compounds.

Underlying thermodynamics of pH-dependent allostery.

Understanding the effects of coupling protein protonation and conformational states is critical to the development of drugs targeting pH sensors and to the rational engineering of pH switches. In this work, we address this issue by performing a comprehensive study of the pH-regulated switch from the closed to the open conformation in nitrophorin 4 (NP4) that determines its pH-dependent activity. Our calculations show that D30 is the only amino acid that has two significantly different pKas in the open and closed conformations, confirming its critical role in regulating pH-dependent behavior. In addition, we describe the free-energy landscape of the conformational change as a function of pH, obtaining accurate estimations of free-energy barriers and equilibrium constants using different methods. The underlying thermodynamic model of the switch workings suggests the possibility of tuning the observed pKa only through the conformational equilibria, keeping the same conformation-specific pKas, as evidenced by the proposed K125L mutant. Moreover, coupling between the protonation and conformational equilibria results in efficient regulation and pH-sensing around physiological pH values only for some combinations of protonation and conformational equilibrium constants, placing constraints on their possible values and leaving a narrow space for protein molecular evolution. The calculations and analysis presented here are of general applicability and provide a guide as to how more complex systems can be studied, offering insight into how pH-regulated allostery works of great value for designing drugs that target pH sensors and for rational engineering of pH switches beyond the common histidine trigger.

Computational study on the structure and properties of ternary complexes of Ln3+ (Ln = La, Ce, Nd AND Sm) with 5,7-dichloroquinoline-8-ol and 4-vinyl pyridine

In the present research, we have mainly concentrated on the survey of interactions in Ln3+ (Ln = La, Ce, Nd, and Sm) ternary complexes of 5,7-dichloroquinoline-8-ol (DCQ) and 4-vinyl pyridine (VP), [Ln(VP)2(DCQ)3]3+ by means of density functional theory, Hartree-Fock and Sparkle/PM3 semi-empirical computational methods. For VP and DCQ ligands, the cation binding energy sequence follows the order La3+ > Ce3+ > Nd3+ > Sm3+ as expected based on increasing in the hardness and decreasing in the ionic radius of this lanthanide cation series. A similar trend was observed in the calculated binding energy of the aforesaid ligands with the hydrated lanthanide cation series [Ln(H2O)9]3+, while the computed values of deformation energy of ligands upon complexation demonstrated an opposite order in the lanthanide cation series. Moreover, the solvent effects are considered via a polarized continuum model and provided a significant increase in the binding strength while the relative magnitude of binding energies is the same as that in the gas phase. Combining quantum and statistical mechanical calculations, we have also determined quantitatively a reliable estimate of the conformational distribution of the [Sm(VP)2(DCQ)3]3+ complex at various temperatures in the gas phase by computing the molecular partition functions and consequently the analysis of the conformational equilibrium constants.

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

Selected‐fit versus induced‐fit protein binding: Kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.

Site-specific Binding Affinities within the H2B Tail Domain Indicate Specific Effects of Lysine Acetylation

Acetylation of specific lysines within the core histone tail domains plays a critical role in regulating chromatin-based activities. However, the structures and interactions of the tail domains and the molecular mechanisms by which acetylation directly alters chromatin structures are not well understood. To address these issues we developed a chemical method to quantitatively determine binding affinities of specific regions within the individual tail domains in model chromatin complexes. Examinations of specific sites within the H2B tail domain indicate that this tail contains distinct structural elements and binds within nucleosomes with affinities that would reduce the activity of tail-binding proteins 10-50-fold from that deduced from peptide binding studies. Moreover, we find that mutations mimicking lysine acetylation do not cause a global weakening of tail-DNA interactions but rather the results suggest that acetylation leads to a much more subtle and specific alteration in tail interactions than has been assumed. In addition, we provide evidence that acetylation at specific sites in the tail is not additive with several events resulting in similar, localized changes in tail binding.

Solvent and substituent effects on the conformational equilibria and intramolecular hydrogen bonding of 4-substituted-2-hydroxybenzaldehydes

By applying the B3LYP/6-31G(d) method with the SCIPCM model on seven 4X substituted 2-hydroxybenzaldehydes, some structural characteristics related with their conformational equilibria and intramolecular hydrogen bonds have been clarified. The compounds are almost completely under the planar conformation characterized by a strong intramolecular hydrogen bond, which decreases in those solvents that possess a higher hydrogen bond donating capability and polarity. The substituents exert a marked influence on the conformational equilibrium constants and the strength of the IHB. Moreover, the excellent Hammett-type equations obtained support the proposed conformational reactions to quantify the IHB in the o-hydroxybenzaldehydes studied.

Stereochemical studies by molecular palpation

AbstractThe conformational equilibrium of cyclohexanol was investigated by 129Xe NMR spectroscopy. While the classical NMR approach focuses on a carbon or proton atom belonging to the molecule under investigation, in our 129Xe NMR methodology we use the xenon atom as an external spy. Xenon is able to monitor the interconversion between the axial and the equatorial isomers of cyclohexanol. The conformational equilibrium constant was estimated and is in excellent agreement with the values obtained by 1H and 13C NMR. Copyright © 2004 John Wiley & Sons, Ltd.

Solvation effects on equilibria: Triazoles and N-methyl piperidinol

We have performed calculations of the solvation effects on a number of equilibrium constants in water using a recently proposed hybrid quantum–classical scheme in which the liquid environment is modelled using classical solvent molecules and the solute electronic structure is computed using modern quantum chemical methods. The liquid phase space is sampled from a fully classical simulation. We find that solvation effects on both triazole tautomeric equilibrium constants and piperidinol conformational equilibrium constants can be interpreted in terms of subtle differences in the local environment which can be seen in probability densities and radial distribution functions. Lower level calculations were performed for comparison and we conclude that the solvation thermodynamics can be predicted from a good classical model of solvent and solute molecules, but the implicit models that we tried are less successful.

Gas-Phase 1H NMR Studies of Internal Rotation Barriers and Conformer Stabilities of N-Ethyl, N-Methylthioamides

Kinetic parameters and conformational equilibrium constants characterizing the internal rotation in gaseous N-ethyl, N-methylthioformamide (EMSF), N-ethyl, N-methylthioacetamide (EMSA), and N-ethyl, N-methyltrifluorothioacetamide (EMSTFA) are determined from exchange broadened 1H NMR spectra. The ΔG⧧298 values, in kcal mol-1, are 22.6 (0.1), 17.8 (0.1), and 17.2(0.1) for EMSF, EMSA, and EMSTFA, respectively. Conformer Gibbs free energy differences, ΔG° (ΔG° = G° (methyl group syn to thionyl sulfur) − G° (methyl group anti to thionyl sulfur)), in cal mol-1, are −123 (24), 56 (9), and 96 (13) for EMSF, EMSA, and EMSTFA, respectively. These results are compared to solution phase values and to the corresponding oxoamides.

Structural analysis of flavonoids with anti-HIV activity

A theoretical study on the structure of twenty flavonoids with anti-HIV activity was performed. The structural properties of these compounds were determined with the AM1 method. Analysis of the obtained quantum data with the Principal Components Analysis (PCA) method showed that the compounds belong to four well differentiated structural patterns: flavones, flavonols, flavanones and benzo-γ-pyrones. In addition, using the Hierarchical Cluster Analysis (HCA) method, the flavonoids were classified into three groups according to their conformational properties. The obtained results permitted to propose a very good relation between the conformational equilibrium constants of these compounds with the structural characteristics of their conformers.

Heterogeneity of kinetics and electron transfer equilibria in the bacteriopheophytin and quinone electron acceptors of reaction centers from Rhodopseudomonas viridis

Heterogeneity of kinetics and electron transfer equilibria in the bacteriopheophytin and quinone electron acceptors of reaction centers from Rhodopseudomonas viridis

Solvent Effects upon the Conformational Equilibrium for the Cofacial Zinc(II) Tetraphenylporphyrin Cage Molecule

Experimental values for conformational equilibrium constants of the zinc(II) tetraphenylporphyrin cage molecule in seventeen nonaqueous solvents have been published recently. That data collection has now been critically re-examined in order to identify the characteristics of the solvent associated with the observed changes in the distribution of the solute between the introverted and extroverted forms of the porphyrin-quinone. Based on a linear solvation energy analysis for a wide range of solvent types, it now appears that the primary influences which are attributable to the solvent environment include the dipolarity, inductive polarizability, and hydrogen bonding by the solvent species. The biochemical metalloporphyrins have been studied extensively as chemical agents in oxygen transfer and storage and in electron transfer processes. A number of synthetic metalloporphyrins have been examined as well in order to isolate and more fully identify particular structural features contributing to photochemical, mechanistic and catalytic properties of their more complex natural analogues. Among these synthetic compounds, zinc(II) tetraphenyl-porphyrin (ZnTPP) and its derivatives have served as model structures for the investigation of the influence of axial ligation on electronic spectra (Nappa and Valentine, 1978) and thermodynamic changes which accompany modifications in the side-chain substituents on the porphyrin (Walker and Benson, 1980). Of the many ZnTPP derivatives, one structurally unique and interesting porphyrin-quinone cage molecule has been recently synthesized and fully characterized by Lisicki et al. (1988). This molecule can most easily be pictured as having an overall hemispherical shape with the base of the hemisphere being the Zn-porphyrin planar structure and the quinone portion located at the polar position above the center of the porphyrin ring. Thus, the quinone portion is attached to the perimeter of the ZnTPP plane by four bridging arms or spacer groups.

Dependence of the conformational equilibrium of alkyl azidoformates on the length of the carbon chain of the alkyl group

An empriical dependence of the conformational equilibrium constant of alkyl azidoformates on the length of the alkyl group carbon chain was derived on the basis of an experimental study.

Electron transfer reactions and associated conformational changes: Electrochemical reduction of trans- 1,2-diiodocyclohexane

The preparation of trans-1,2-diiodocyclohexane by reaction of cyclohexene and iodine has been characterized. Equilibrium favors the diiodide in chloroform or cyclohexene but dissociation occurs in dimethylformamide (DMF). Solutions in cyclohexene were introduced into cold DMF for study in the latter solvent. 1H and 13C NMR were used to confirm that the trans isomer is formed and to obtain relative populations of the conformers with axial iodines (aa) and equatorial iodines (ee). The ratio of concentrations of aa to ee is 4.0 in DMF at −55° C. Platinum microdisc electrodes were used to investigate the electrochemical reduction by cyclic voltammetry. At low temperatures and high scan rates in DMF solvent, the reduction peak for trans-1,2-diiodocyclohexane splits into two peaks. The peak appearing at less negative potential was assigned to the reduction of aa and the second peak to ee. Digital simulations were fit to the background-corrected voltammograms to obtain the conformational equilibrium and rate constants for temperatures in the range of 0 to −40° C.

A nuclear magnetic resonance study of the formation and conformational equilibria of symmetrical and mixed disulfides of captopril

The oxidation of captopril (CpSH, 1-(D-3-mercapto-2-methylpropanoyl)-1-proline) by glutathione disulfide (GSSG) via thiol/disulfide exchange to form, in the first step, CpSSG and GSH and, in the second step, CpSSCp and GSH, has been studied in aqueous solution by 1H nmr. Due to slow rotation around the amide bond(s) of CpSH and CpSSCp and of the captopril part of CpSSG, separate resonances are observed for the cis and trans conformations across these bonds. Conformational equilibrium constants were estimated as a function of pH for CpSH, CpSSCp, and CpSSG from the intensities of resonances for the cis and trans isomers. These equilibrium constants were used in the determination of equilibrium constants for the two steps in the oxidation of CpSH by GSSG. The results suggest that CpSH has a greater tendency to reduce disulfide bonds by thiol/disulfide exchange at physiological pH, and thus form mixed disulfides, than do the thiol groups in amino acids. Also, the conformational equilibrium constants indicate that, at physiological pH, approximately two thirds of the captopril, either free or in a disulfide form, has the trans conformation.

Conformational analysis of β-D-ribo-, β-D-deoxyribo-, β-D-arabino-, β-D-xylo-, and β-D-lyxo-nucleosides from proton–proton coupling constants

A new n.m.r. coupling constant torsion angle relation is utilized to explore the effect of exocyclic oxygen substituents at C-2′ and C-3′ on J1′2.′, J2′,3′, and J3′,4′ in the furanose ring of β-D-ribo-, β-D-arabino-, β-D-xylo-, and β-D-lyxonucleosides. The five vicinal couplings in β-D-deoxyribonucleosides are also investigated. Calculated coupling constants for the full pseudorotational itinerary at different values of the puckering amplitudes are given. It is shown that experimental coupling constants, taken from the literature, can be satisfactorily explained in all cases on the basis of a two-state (N/S)-model of the furanose pseudorotation. Good estimates of both N- and S- geometry and the conformational equilibrium constant in five-membered rings of ribose, deoxyribose, xylose, lyxose, and arabinose derivatives are given. The judicious use of geometrical information obtained by X-ray crystallographic studies appeared indispensable for this type of analysis.

Effect of lanthanide shift reagents on conformational equilibria

AbstractThe conformational equilibrium constants for a series of 1‐alkyl substituted cyclohexanols have been determined using a lanthanide shift reagent, and the results compared with other methods. In comparison to other technique greater preference is shown by the hydroxyl group for the equatorial position. This is rationalized by the effect on the equilibrium of the bulky shift reagent.

Hydroxyl‐Ion‐Induced Subunit Dissociation of Yeast Cytoplasmic Pyruvate Decarboxylase

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.