Changes In Conformational Equilibrium Research Articles

Synthesis and spectroscopic characterization of thioparacetamol: Evidence for solvent-dependent changes in conformational equilibrium

The search for analogs of compounds with proved pharmacological activity frequently starts by obtaining those that result from simple chemical substitutions, have structural and mechanical characteristics suitable for inclusion in a formulation and close similarity with related lead compounds. With this aim, the isosteric replacement of the carbonyl functional group of paracetamol to obtain solid thioparacetamol was achieved through microwave mediated thionation of parent compound in very good yield (85 %). A thorough characterization of the compound prepared was performed by GC/MS spectrometry, FTIR and NMR (1H, 13C, COSY, HSQC and HMBC) spectroscopies and quantum chemical calculations derived from density functional theory. From the experimental and theoretical spectra, it was evidenced that the structural, vibrational and conformational properties of this compound vary whether the sample is solved in a solvent or not. The anti form (anti of the CS and NH bonds) was the preferred conformational isomer in the solid phase, while a conformational equilibrium of anti and syn forms was found for samples solved in different solvents. As polarity of solvents increased, proportion of the anti isomer increased as well.

Hidden dynamic signatures drive substrate selectivity in the disordered phosphoproteome

Phosphorylation sites are hyperabundant in the eukaryotic disordered proteome, suggesting that conformational fluctuations play a major role in determining to what extent a kinase interacts with a particular substrate. In biophysical terms, substrate selectivity may be determined not just by the structural-chemical complementarity between the kinase and its protein substrates but also by the free energy difference between the conformational ensembles that are, or are not, recognized by the kinase. To test this hypothesis, we developed a statistical-thermodynamics-based informatics framework, which allows us to probe for the contribution of equilibrium fluctuations to phosphorylation, as evaluated by the ability to predict Ser/Thr/Tyr phosphorylation sites in the disordered proteome. Essential to this framework is a decomposition of substrate sequence information into two types: vertical information encoding conserved kinase specificity motifs and horizontal information encoding substrate conformational equilibrium that is embedded, but often not apparent, within position-specific conservation patterns. We find not only that conformational fluctuations play a major role but also that they are the dominant contribution to substrate selectivity. In fact, the main substrate classifier distinguishing selectivity is the magnitude of change in local compaction of the disordered chain upon phosphorylation of these mostly singly phosphorylated sites. In addition to providing fundamental insights into the consequences of phosphorylation across the proteome, our approach provides a statistical-thermodynamic strategy for partitioning any sequence-based search into contributions from structural-chemical complementarity and those from changes in conformational equilibrium.

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy.

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5' cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA.

Perspectives on electrostatics and conformational motions in enzyme catalysis.

ConspectusEnzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR).We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH.Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Universal buffers for use in biochemistry and biophysical experiments.

The use of buffers that mimic biological solutions is a foundation of biochemical and biophysical studies. However, buffering agents have both specific and nonspecific interactions with proteins. Buffer molecules can induce changes in conformational equilibria, dynamic behavior, and catalytic properties merely by their presence in solution. This effect is of concern because many of the standard experiments used to investigate protein structure and function involve changing solution conditions such as pH and/or temperature. In experiments in which pH is varied, it is common practice to switch buffering agents so that the pH is within the working range of the weak acid and conjugate base. If multiple buffers are used, it is not always possible to decouple buffer induced change from pH or temperature induced change. We have developed a series of mixed biological buffers for protein analysis that can be used across a broad pH range, are compatible with biologically relevant metal ions, and avoid complications that may arise from changing the small molecule composition of buffers when pH is used as an experimental variable.

Hydrogen Tunneling, Electrostatics, and Conformational Motions in Enzyme Catalysis

The roles of hydrogen tunneling, electrostatics, and conformational motions in enzyme catalysis will be discussed. We have developed hybrid quantum/classical molecular dynamics methods that include the quantum mechanical effects of the active electrons and transferring proton(s), as well as the motions of the entire solvated enzyme. These methods have been used to study the proton and hydride transfer reactions catalyzed by the enzymes dihydrofolate reductase (DHFR) and ketosteroid isomerase (KSI). The free energy profiles are generated along a collective reaction coordinate, and the changes in hydrogen bonding and electrostatic interactions are analyzed along the entire reaction pathway. An analysis of the simulations resulted in the identification and characterization of a network of coupled motions that extends throughout the enzyme and represents equilibrium conformational changes that facilitate the chemical reaction. Mutations distal to the active site are shown to significantly impact the catalytic rate constant by altering the conformational sampling of the entire enzyme, thereby changing the probability of sampling configurations conducive to the catalyzed reaction. We have also developed quantum mechanical/molecular mechanical methodology to calculate the vibrational frequency shifts of thiocyanate probes incorporated into the active site of an enzyme. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of an intermediate analog to KSI for two different nitrile probe positions. Analysis of the simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. This approach is also being used to study the vibrational shifts of nitrile probes for intermediates along the reaction pathway of DHFR to elucidate the conformational changes and the variation of the electrostatic microenvironments during catalysis.

Catalytic Efficiency of Enzymes: A Theoretical Analysis

This brief review analyzes the underlying physical principles of enzyme catalysis, with an emphasis on the role of equilibrium enzyme motions and conformational sampling. The concepts are developed in the context of three representative systems, namely, dihydrofolate reductase, ketosteroid isomerase, and soybean lipoxygenase. All of these reactions involve hydrogen transfer, but many of the concepts discussed are more generally applicable. The factors that are analyzed in this review include hydrogen tunneling, proton donor-acceptor motion, hydrogen bonding, pKa shifting, electrostatics, preorganization, reorganization, and conformational motions. The rate constant for the chemical step is determined primarily by the free energy barrier, which is related to the probability of sampling configurations conducive to the chemical reaction. According to this perspective, stochastic thermal motions lead to equilibrium conformational changes in the enzyme and ligands that result in configurations favorable for the breaking and forming of chemical bonds. For proton, hydride, and proton-coupled electron transfer reactions, typically the donor and acceptor become closer to facilitate the transfer. The impact of mutations on the catalytic rate constants can be explained in terms of the factors enumerated above. In particular, distal mutations can alter the conformational motions of the enzyme and therefore the probability of sampling configurations conducive to the chemical reaction. Methods such as vibrational Stark spectroscopy, in which environmentally sensitive probes are introduced site-specifically into the enzyme, provide further insight into these aspects of enzyme catalysis through a combination of experiments and theoretical calculations.

Thermodynamics and kinetics of large‐time‐step molecular dynamics

Molecular dynamics (MD) simulations provide essential information about the thermodynamics and kinetics of proteins. Technological advances in both hardware and algorithms have seen this method accessing timescales that used to be unreachable only few years ago. The quest to simulate slow, biologically relevant macromolecular conformational changes, is still open. Here, we present an approximate approach to increase the speed of MD simulations by a factor of ∼4.5. This is achieved by using a large integration time step of 7 fs, in combination with frozen covalent bonds and look-up tables for nonbonded interactions of the solvent. Extensive atomistic MD simulations for a flexible peptide in water show that the approach reproduces the peptide's equilibrium conformational changes, preserving the essential properties of both thermodynamics and kinetics. Comparison of this approximate method with state-of-the-art implicit solvation simulations indicates that the former provides a better description of the underlying free-energy surface. Finally, simulations of a 33-residue peptide show that these fast MD settings are readily applicable to investigate biologically relevant systems.

Structural Diversity and Changes in Conformational Equilibria of Biantennary Complex-Type N-Glycans in Water Revealed by Replica-Exchange Molecular Dynamics Simulation

Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain “key” (or group of “keys”) within the framework of the “bunch of keys” mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification.

Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while NH(2)-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the NH(2)-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells.

PTH Receptor Responds To Shear Stress Perturbation of Plasma Membrane

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).This research was supported by the NIH grant HL086943.

Relating Protein Motion to Catalysis

This review examines the linkage between protein conformational motions and enzyme catalysis. The fundamental issues related to this linkage are probed in the context of two enzymes that catalyze hydride transfer, namely dihydrofolate reductase and liver alcohol dehydrogenase. The extensive experimental and theoretical studies addressing the role of protein conformational changes in these enzyme reactions are summarized. Evidence is presented for a network of coupled motions throughout the protein fold that facilitate the chemical reaction. This network is comprised of fast thermal motions that are in equilibrium as the reaction progresses along the reaction coordinate and that lead to slower equilibrium conformational changes conducive to the chemical reaction.

Freezing a Single Distal Motion in Dihydrofolate Reductase

Constraining a single motion between distal residues separated by approximately 28 A in hybrid quantum/classical molecular dynamics simulations is found to increase the free energy barrier for hydride transfer in dihydrofolate reductase by approximately 3 kcal/mol. Our analysis indicates that a single distal constraint alters equilibrium motions throughout the enzyme on a wide range of time scales. This alteration of the conformational sampling of the entire system is sufficient to significantly increase the free energy barrier and decrease the rate of hydride transfer. Despite the changes in conformational sampling introduced by the constraint, the system assumes a similar transition state conformation with a donor-acceptor distance of approximately 2.72 A to enable the hydride transfer reaction. The modified thermal sampling leads to a substantial increase in the average donor-acceptor distance for the reactant state, however, thereby decreasing the probability of sampling the transition state conformations with the shorter distances required for hydride transfer. These simulations indicate that fast thermal fluctuations of the enzyme, substrate, and cofactor lead to conformational sampling of configurations that facilitate hydride transfer. The fast thermal motions are in equilibrium as the reaction progresses along the collective reaction coordinate, and the overall average equilibrium conformational changes occur on the slower time scale measured experimentally. Recent single molecule experiments suggest that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time scale of the hydride transfer reaction. Thus, introducing a constraint that modifies the conformational sampling of an enzyme could significantly impact its catalytic activity.

Osmolyte-induced changes in protein conformational equilibria.

Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria.

A C-Terminal Conformational Equilibrium in Thymidylate Synthase Observed by Electron Paramagnetic Resonance Spectroscopy

A spin-label was attached to the C-terminal side chain of Lactobacillus casei thymidylate synthase (TS, EC2.1.1.45), and EPR spectroscopy was used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate analog CB3717. The C244T/V316C mutant TS has only two cysteines, the active site Cys-198 and an engineered cysteine which replaces valine as the C-terminal residue. dUMP was used to block the active-site cysteine while the C-terminus was reacted with the spin-label 4-maleimido-2,2,6,6- tetramethylpiperidinyl-1-oxy. Exclusive attachment of the label to the C-terminal cysteine was verified by a study of the labeled enzyme's reaction with 5,5'-dithiobis(2-nitrobenzoic acid). EPR spectra of the labeled enzyme and its complexes were composed of two components corresponding to populations of both flexible and more immobilized forms of the C-terminus (tau C = 1 and 9.7 ns, respectively). Ligand binding increased the population of the more immobilized form of the C-terminus with the following series: free enzyme < E.dUMP approximately dTMP approximately E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced perturbation of the conformational equilibrium was titratable and indicated approximate Kd values of 3 and 13 microM for formation of the E.dUMP and E.CB3717 binary complexes, respectively, and 7 microM for the binding of CB3717 to the E.dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal residue in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the position of the equilibrium changes in the presence of ligands.

The Investigation of Benzene Influence on Conformational Equilibrium in a Solvent Using the Spectral Shift Method

Abstract A consequence of an improved model of dissolution process passing through the cavity creation stage is discussed. The improvement consists in accounting for the scales of initial density fluctuations (appropriate vacancies) necessary for creation of the cavities. The radii (R 1u v ) of the cavities in solvents with flexible molecules containing benzene as a solute, evaluated from solvent-induced spectral shifts, and radii of the vacancies, calculated from volume balance involving the R 1u v 's, have been used for determining the changes in conformational equilibrium in the solvents caused by the solute. In free energy balance calculations performed with this aim, the free energy of a cavity is estimated according to Sinanoglu, when the cavity size is equal to or exceeds the size of the room occupied by a solvent molecule, but in the submolecular region it is expressed in terms of the probability of accidental collision of little cavity defects. The reasonable values obtained confirm the picture of dissolution deduced from spectroscopy data.

ChemInform Abstract: EFFECT OF THE SOLVENT‐DEPENDENT CONFORMATIONAL SYSTEM OF HYDROXYUREAS ON PREDICTED VS. OBSERVED LOG P

Calculated and observed log P values are reported and compared with in vivo and in vitro biological action (L1210 leukemia ILS % and ribonucleotide reductase ID50) for hydroxyurea, the 1-N methyl and ethyl, and the 3-N ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, phenyl, and p-chlorophenyl analogues. The log P values were calculated via the method of Hansch and Leo from literature f values and the observed log P values were obtained by direct determination after equilibration between octanol and water. Calculations of log P for hydroxyurea were found to be appreciably more hydrophilic than the values obtained experimentally. Differences in calculated and observed log P (delta log P) for the substituted analogues were lowest with the 1-N and the bulky 3-N substituents and greatest with the 3-N-substituted straight-chain analogues (delta log P = 0.70). Different structural species were observed by infrared spectroscopy in dry octanol vs. octanol after water equilibration and drying, and this is proposed as due to changes in conformational equilibrium in the hydroxyurea systems. Differences between calculated and observed log P are explained via the stabilization of internally hydrogen-bonded conformers in the case of 1-N or bulky 3-N analogues or destabilization of various conformers allowing maximal interactions with solvent or water which is the case with straight chain 3-N analogues.

Effects of unshared pairs of electrons and their solvation on conformational equilibria

Abstract

Steroidal sulphur compounds. Part V. Configuration, conformation, and chiroptical properties of steroidal benzyl sulphoxides

Steroidal C-3 and C-6 benzyl sulphoxides of (R)-configuration at sulphur displayed negative Cotton effects below 220 nm. and positive Cotton effects in the range 229–236 nm. in ethanol. The Cotton effects are of greater amplitude and occur at longer wavelengths than those of the corresponding methyl sulphoxides, in analogy with the relation between dialkyl and alkyl benzyl ketones. Changes in conformational equilibria were probably not important in solvent-induced variations in amplitudes of the Cotton effects. Pyrolysis of (R)-3α-benzylsulphinyl-5α-cholestane gave 5α-cholest-2-ene regiospecifically.