Conformer Populations Research Articles

Refinement of Peptide Conformational Ensembles by 2D IR Spectroscopy: Application to Ala‒Ala‒Ala

Characterizing ensembles of intrinsically disordered proteins is experimentally challenging because of the ill-conditioned nature of ensemble determination with limited data and the intrinsic fast dynamics of the conformational ensemble. Amide I two-dimensional infrared (2D IR) spectroscopy has picosecond time resolution to freeze structural ensembles as needed for probing disordered-protein ensembles and conformational dynamics. Also, developments in amide I computational spectroscopy now allow a quantitative and direct prediction of amide I spectra based on conformational distributions drawn from molecular dynamics simulations, providing a route to ensemble refinement against experimental spectra. We performed a Bayesian ensemble refinement method on Ala–Ala–Ala against isotope-edited Fourier-transform infrared spectroscopy and 2D IR spectroscopy and tested potential factors affecting the quality of ensemble refinements. We found that isotope-edited 2D IR spectroscopy provides a stringent constraint on Ala–Ala–Ala conformations and returns consistent conformational ensembles with the dominant ppII conformer across varying prior distributions from many molecular dynamics force fields and water models. The dominant factor influencing ensemble refinements is the systematic frequency uncertainty from spectroscopic maps. However, the uncertainty of conformer populations can be significantly reduced by incorporating 2D IR spectra in addition to traditional Fourier-transform infrared spectra. Bayesian ensemble refinement against isotope-edited 2D IR spectroscopy thus provides a route to probe equilibrium-complex protein ensembles and potentially nonequilibrium conformational dynamics.

Solvation of Mg2+ Ions in Mg(TFSI)2–Dimethoxyethane Electrolytes—A View from Molecular Dynamics Simulations

Classical, polarizable molecular dynamics simulations have been performed for a series of Mg(N(SO2CF3)2)2–dimethoxyethane electrolytes with salt concentration varying in the range c = 0.1–1.2 M. It has been found that in dimethoxyethane solutions, magnesium salt exists as free ions, with metal cations coordinated to solvent molecules. Cation–solvent interactions favor TTT, TGT, and TGG′ conformations of dimethoxyethane, modifying the population of conformers in the electrolyte. Mg2+ ions form stable solvates with about three dimethoxyethane molecules. Two sets of solvent molecules with different mobility have been detected in simulations: fast diffusing free solvent molecules and dimethoxyethane molecules bound in the solvates, diffusing slowly with magnesium ions. All these findings are in good agreement with the recent experimental data.

Dissecting the Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET.

Conformational changes of enzyme proteins are often coupled with a catalytic reaction and modulate the enzyme activity. Single-molecule technology is a powerful tool to study the mechanism of enzyme catalysis in these complicated cases. However, the chemical reaction cycles and conformational changes could not be monitored simultaneously in a single-molecule detection experiment, resulting in some unresolved key kinetic parameters. Here, we describe a method to extract all of the kinetic parameters from comprehensive single-molecule FRET (smFRET) measurements and model analysis. On the basis of the smFRET, we calculated the undetectable parameters by solving the rate equations of the kinetic model with the input of the smFRET-measured conformational state populations and state-transition rate constants. A case study of MalK2 ATPase demonstrates that this method could reveal the quantitative mechanism of the catalytic reaction of the enzyme as well as its coupled conformational dynamics. The strategy employed in this study could be widely applied to investigate the conformational fluctuation-coupled catalysis of other enzymes.

Structural, spectroscopic and thermodynamic aspects of azide–water clusters: an approach using a conjugated prescription of stochastic and quantum chemical methods

ABSTRACTIn this work, we have used the stochastic search strategy of adaptive mutation simulated annealing (AMSA) as the search tool to explore quantum chemical potential energy to find structures for clusters for n up to 8. The evaluated structures are then further refined at higher levels of theories to get more accurate values of IR frequencies, interaction energy (IE), solvation energy (SE) and vertical detachment energy (VDE). The variations of IE, SE and VDE are also observed and critically discussed to understand the observed trends and their correspondence with experimental findings. In a real experimental scenario, where finite temperature is present, any obtained property will be dictated by contribution from local isomers along with the global one. Keeping this important fact in mind, we have investigated conformational populations of different isomers for the atmospherically relevant temperature range between 200 K and 300 K.

Defining a conformational ensemble that directs activation of PPAR\u03b3

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Here, we present a nuclear receptor co-regulator-binding surface structural ensemble in solution, viewed through the lens of fluorine-19 (19F) nuclear magnetic resonance (NMR) and molecular simulations, and the response of this ensemble to ligands, co-regulator peptides and heterodimerization. We correlate the composition of this ensemble with function in peroxisome proliferator-activated receptor-γ (PPARγ) utilizing ligands of diverse efficacy in co-regulator recruitment. While the co-regulator surface of apo PPARγ and partial-agonist-bound PPARγ is characterized by multiple thermodynamically accessible conformations, the full and inverse-agonist-bound PPARγ co-regulator surface is restricted to a few conformations which favor coactivator or corepressor binding, respectively.

Conformational study of melamine crosslinkers and spectroscopical comparison of HMMM molecules by practical measurements and quantum chemical calculations

To understand and improve network formation processes and performance properties makes structural molecular studies critically important for the coil-coating industry. Crosslinking agents such as hexamethoxymethylmelamine (HMMM) or less methylated derivatives, e.g. methoxymethylmelamine (MMM), are often added to industrial coating formulations. For molecules with considerably fewer atoms and accordingly less rotational freedom (such as MMM) it is readily accessible to identify principal conformations. Thus, an MMM conformer study is straightforward and serves as orientation concerning HMMM conformer studies. For HMMM molecules an extensive computation method was developed to investigate the conformational distribution average and probably most likely molecular structures. Using the density functional theory (DFT) B3LYP-D3BJ method with Dunning's correlation basis set, calculations were performed to investigate the three-dimensional structural geometries of HMMM (basis set cc-pVDZ) and MMM (basis set cc-pVTZ). Beginning with 1500 conformations for HMMM and using various cut-off filters we focused on final residual 22 conformers for solvent phase calculations and 16 conformers for gas phase calculations. To the best of our knowledge, this is the first time that chemical properties for melamine crosslinkers were presented under consideration of conformational population distribution. Thus, computations of fully optimized structural geometries, energies and vibrational states indicate that preferred structural alignments for the methoxymethyl (MM) group in melamine molecules exist. Nevertheless, we suppose that as a matter of principle the MM group of MMM molecules can easily perform rotations by itself and all conformational structure geometries of MMM will exist. Concerning the MM groups position compared to triazine plane HMMM molecules have two main configurations,”3up-3down” and “4up-2down”, as dominant conformer species. Computations of weighted and averaged IR and Raman spectra of final conformations for HMMM monomers, dimers and trimers at ambient temperature are novel and in good agreement with experiment. Prediction of a final UV–Vis spectrum of top ten Boltzmann-weight and averaged monomeric HMMM conformers is in perfect agreement with practical measurement of commercially available HMMM.

Spectra-structure correlations in NIR region: Spectroscopic and anharmonic DFT study of n-hexanol, cyclohexanol and phenol

We investigated near-infrared (7500–4000 cm−1) spectra of n-hexanol, cyclohexanol and phenol in CCl4 (0.2 M) by using anharmonic quantum calculations. These molecules represent three major kinds of alcohols; linear and cyclic aliphatic, and aromatic ones. Vibrational second-order perturbation theory (VPT2) was employed to calculate the first overtones and binary combination modes and to reproduce the experimental NIR spectra. The level of conformational flexibility of these three alcohols varies from one stable conformer of phenol through four conformers of cyclohexanol to few hundreds conformers in the case of n-hexanol. To take into account the most relevant conformational population of n-hexanol, a systematic conformational search was performed. Accurate reproduction of the experimental NIR spectra was achieved and detailed spectra-structure correlations were obtained for these three alcohols. VPT2 approach provides less reliable description of highly anharmonic modes, i.e. OH stretching. In the present work this limitation was manifested in erroneous results yielded by VPT2 for 2νOH mode of cyclohexanol. To study the anharmonicity of this mode we solved the corresponding time-independent Schrödinger equation based on a dense-grid probing of the relevant vibrational potential. These results allowed for significant improvement of the agreement between the calculated and experimental 2νOH band of cyclohexanol. Various important biomolecules include similar structural units to the systems investigated here. A detailed knowledge on spectral properties of these three types of alcohols is therefore essential for advancing our understanding of NIR spectroscopy of biomolecules.

Unveiling the pathway to Z-DNA in the protein-induced B–Z transition

Left-handed Z-DNA is an extraordinary conformation of DNA, which can form by special sequences under specific biological, chemical or physical conditions. Human ADAR1, prototypic Z-DNA binding protein (ZBP), binds to Z-DNA with high affinity. Utilizing single-molecule FRET assays for Z-DNA forming sequences embedded in a long inactive DNA, we measure thermodynamic populations of ADAR1-bound DNA conformations in both GC and TG repeat sequences. Based on a statistical physics model, we determined quantitatively the affinities of ADAR1 to both Z-form and B-form of these sequences. We also reported what pathways it takes to induce the B–Z transition in those sequences. Due to the high junction energy, an intermediate B* state has to accumulate prior to the B–Z transition. Our study showing the stable B* state supports the active picture for the protein-induced B–Z transition that occurs under a physiological setting.

Model Selection Using BICePs: A Bayesian Approach for Force Field Validation and Parameterization.

The Bayesian Inference of Conformational Populations (BICePs) algorithm reconciles theoretical predictions of conformational state populations with sparse and/or noisy experimental measurements. Among its key advantages is its ability to perform objective model selection through a quantity we call the BICePs score, which reflects the integrated posterior evidence in favor of a given model, computed through free energy estimation methods. Here, we explore how the BICePs score can be used for force field validation and parametrization. Using a 2D lattice protein as a toy model, we demonstrate that BICePs is able to select the correct value of an interaction energy parameter given ensemble-averaged experimental distance measurements. We show that if conformational states are sufficiently fine-grained, the results are robust to experimental noise and measurement sparsity. Using these insights, we apply BICePs to perform force field evaluations for all-atom simulations of designed β-hairpin peptides against experimental NMR chemical shift measurements. These tests suggest that BICePs scores can be used for model selection in the context of all-atom simulations. We expect this approach to be particularly useful for the computational foldamer design as a tool for improving general-purpose force fields given sparse experimental measurements.

Infrared Population Transfer Spectroscopy of Cryo-Cooled Ions: Quantitative Tests of the Effects of Collisional Cooling on the Room Temperature Conformer Populations.

The single-conformation spectroscopy and infrared-induced conformational isomerization of a model protonated pentapeptide [YGPAA + H]+ is studied under cryo-cooled conditions in the gas phase. Building on recent results ( DeBlase , A. F. ; J. Am. Chem. Soc. 2017 , 139 , 5481 - 5493 ), firm assignments are established for the presence of two conformer families with distinct infrared and ultraviolet spectra, using IR-UV depletion spectroscopy. Families (A and B) share a similar structure near the N-terminus but differ in the way that the C-terminal COOH group configures itself (cis versus trans) in forming H-bonds with the peptide backbone. Infrared population transfer (IR-PT) spectroscopy is used to study the IR-induced conformational isomerization following single-conformer infrared excitation. IR-induced isomerization is accomplished in both directions (A → B and B → A) in the hydride stretch region and is used to determine fractional abundances for the two conformer families (FA = 0.65 ± 0.04, FB = 0.35 ± 0.04, 2σ error bars). The time scale for collisional cooling of the room-temperature ions to Tvib = 10 K by cold helium in the octupole trap is established as 1.0 ms. Key stationary points on the isomerization potential energy surface are calculated at the DFT B3LYP/6-31+G(d) G3DBJ level of theory. Using RRKM theory, the energy-dependent isomerization rates and populations are calculated as a function of energy. According to the model, the observed population distribution after collisional cooling is close to that of the 298 K Boltzmann distribution and is in near-quantitative agreement with experiment. On the basis of this success, inferences are drawn for the circumstances that govern the population distribution in the trap, concluding that, in ions the size of [YGPAA + H]+ and larger, the observed distributions will be near those at 298 K.

Heparin and Heparan Sulfate Binding of the Antiparasitic Drug Imidocarb: Circular Dichroism Spectroscopy, Isothermal Titration Calorimetry, and Computational Studies.

This study is aimed to assess the binding interaction between the antiparasitic cationic drug imidocarb (IMD) and sulfated glycosaminoglycans (GAGs), the ubiquitious nonprotein macromolecules of living organisms. These complex, heterogeneous polyanions are the integral constituents of cell membranes and the extracellular matrix and display affinity toward basic compounds, the binding of which may affect their biological functions. Exciton-type circular dichroism (CD) spectroscopic features measured at low salt concentration verify the heparin and heparan sulfate binding of IMD, which occurs in a cooperative manner by association of several drug molecules to a disaccharide unit. Isothermal titration calorimetry (ITC) measurements reassured the heparin interaction, resulting in a Kd value in the low micromolar range. In contrast, when considering high molar excess of the heparin-binding sites, closer resembling in vivo conditions, an entirely different CD signature was induced, suggesting a shift from the oligo- to monomeric binding mode. This observation was also supported by ITC measurements using an identical sample setup. To better mimic in vivo conditions, several measurements were performed in physiological salt concentration ranges. On the basis of these, the inter- and intramolecular origin of CD activity observed under low- and high-salt conditions refer to electrostatically held oligomeric and intermolecular H-bonded monomeric drug-GAG adducts, respectively. To complement the experimental data, quantum chemical calculations were performed to assess the photophysical and conformational properties of IMD, indicating the existence of nonlinear, nonplanar interconverting conformer populations. Such a structural flexibility may be important in the multiple, cooperative binding of IMD to sterically adjacent GAG sites.

Structure and Dynamics of the CheY Response Regulators from Rhodobacter sphaeroides

The chemotaxis signalling network of E. coli. depends on autophosphorylation of a histidine protein kinase (HPK) in response to a signal from a sensor domain, with subsequent transfer of the phosphoryl group to an aspartate on response regulator (RR) proteins that bind to the flagellar motor and alter its rotation. CheY is a 14kDa single domain RR that is conserved across motile species. It is formed by 5 α-helices and 5 β-strands surrounding a conserved phosphoryl accepting aspartate residue, and once phosphorylated diffuses to the flagellar motor, binding to its FliM component to cause switching of rotational direction. The photosynthetic bacterium Rhodobacter sphaeroides has multiple chemosensory pathways formed by homologues of the E. coli chemosensory proteins. It has six CheY homologues with different effects on chemotaxis. Only CheY6 is able to stop the flagellar motor but either CheY3 or CheY4 are also required for chemotaxis. NMR and computational methods have been used to answer questions about the structure, dynamics and function of two of the CheY's, CheY3 and CheY6. NOEs, chemical shifts and residual dipolar couplings are used to define the structures of CheY3 and CheY6 in their inactive and active states, where phosphorylation is mimicked using BeF3-. We have investigated fast timescale backbone dynamics using the {1H}-15N heteronuclear NOE and have used CPMG relaxation dispersion experiments to detect low populations of alternative conformations. CheY6 differs from the other R. sphaeroides CheYs and E. coli CheY by the insertion of a ten-residue loop before the C-terminal helix. We have deleted this loop region from CheY6 in order to determine, using in vivo and in vitro assays, if it plays a role in the unique function of CheY6 in R. sphaeroides.

Characterization of the Aggregation-Prone Ensemble of Tau in the Presence of Polyphosphates

Tau is a microtubule associated protein implicated in Alzheimer's disease through its aggregation and deposition as neurofibrillary tangles. Tau is both an intrinsically disordered protein and it aggregates very slowly in vitro making it challenging to study the mechanism of aggregation. Typically highly anionic molecular aggregation inducers, such as heparin, are used to induce aggregation in vitro, although these molecules are unlikely to have much physiological relevance. Recently, polyphosphates, linear chain of phosphate residues found in all prokaryotic and eukaryotic cells, have been proposed to act as a universal scaffold for the initiation of aggregation of amyloidogenic proteins. Polyphosphates have been shown to both increase the rate of amyloid fiber formation and to stabilize the fibers. Here, we use single molecule FRET to characterize conformational changes in tau in the presence of polyphosphates. The low concentrations (pM) of protein utilized in single molecule measurements allow for the population of aggregation-prone conformations, while inhibiting aggregation. In the presence of polyphosphates, tau undergoes domain specific conformational changes in which there is an overall expansion of the protein with a concomitant compaction of the microtubule binding region. Conservation of conformational shifts in the presence of different aggregation inducers will provide a model of the consensus conformational features that potentiate tau for aggregation in disease.

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

Modulations of CA2+ Sensitizers and Phosphorylation of cTnT in Dynamic Equilibrium of cTnC N-Domain

An increasing body of evidence suggests cTnC exists in dynamic conformational equilibrium and that Ca2+ binding regulates this equilibrium. However, little direct evidence has been offered to support these claims. The present study aims to directly monitor dynamic conformational equilibrium of cTnC and investigate how the dynamic equilibrium is modulated by the presence of cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+ sensitizers, and a Ca2+ desensitizing phosphomimic of cTnT (cTnT(T204E). To achieve the objective, a dye homodimerization approach is developed and implemented which allows for determination of the dynamic equilibrium between open and closed conformations in cTnC's hydrophobic cleft. Modulation of this equilibrium by other troponin subunits, Ca2+-sensitizers, and phosphomimic of cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+, which increased significantly upon addition of saturating levels of Ca2+. This suggests the Ca2+ induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. Inclusion of cTnI increased the population of open cTnC while inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cTnT(wt), while Ca2+-sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between open and closed conformations of cTnC's hydrophobic cleft are play a significant role in tuning the Ca2+ sensitivity of the heart.

Reconciling Simulated Ensembles of Apomyoglobin with Experimental HDX Data

Hydrogen/deuterium exchange (HDX) is a powerful tool to investigate protein conformational dynamics at equilibrium. In the classic model of HDX, each of the amide hydrogens of a protein can either exist in an “open” conformation, where HDX can occur with the exchange rate kint, or in a “closed” conformation, where HDX rate, kex, gets much slower. The extent of protection for a residue i, is characterized by a protection factor, PFi = kex,i/kint,i, which is related to the effective free energy difference between open and closed states, ΔG, through ln(PFi ) = βΔGi. However, it has been challenging to make direct connections between molecular simulations of native-state protein dynamics, both in predicting HDX PF from trajectory data, and in using experimental protection factors as restraints in simulated ensembles. In this study, we propose an expression, ln(PF)= βc<Nc> + βh<Nh> + β0, to predict the protection factors based on the average number of heavy-atom contacts <Nc> and hydrogen bonds <Nh> observed in simulations. The model parameters are trained on ultra-long, all-atom trajectories of proteins with experimentally measured protection factors (BPTI and ubiquitin). Using Bayesian inference to propagate the uncertainties in model parameters, we then use this model to refine conformational ensembles of apomyoglobin at neutral pH. To minimize bias, we simulated apomyoglobin using a range of weak to strong amide exposure restraints. We applied a transition-based reweighting method, TRAM, to construct a Multi-ensemble Markov model from the combined simulation data, and then used our Bayesian Inference of Conformational Populations (BICePs) algorithm to refine a conformational ensemble that best matched the experimental structural data. The resulting ensemble shows a partially-disordered F/H helix, and atomically detailed information that was previously missing from 2D NMR studies.

Oligosaccharide model of the vascular endothelial glycocalyx in physiological flow

Experiments have consistently revealed the pivotal role of the endothelial glycocalyx layer in vasoregulation and the layer’s contribution to mechanotransduction pathways. However, the exact mechanism by which the glycocalyx mediates fluid shear stress remains elusive. This study employs atomic-scale molecular simulations with the aim of investigating the conformational and orientation properties of highly flexible oligosaccharide components of the glycocalyx and their suitability as transduction molecules under hydrodynamic loading. Fluid flow was shown to have nearly no effect on the conformation populations explored by the oligosaccharide, in comparison with static (diffusion) conditions. However, the glycan exhibited a significant orientation change, when compared to simple diffusion, aligning itself with the flow direction. It is the tethered end of the glycan, an asparagine amino acid, which experienced conformational changes as a result of this flow-induced bias. Our results suggest that shear flow through the layer can have an impact on the conformational properties of saccharide-decorated transmembrane proteins, thus acting as a mechanosensor.

Consensus Conformations of Dinucleoside Monophosphates Described with Well-Converged Molecular Dynamics Simulations.

Dinucleoside monophosphates (DNMPs) have been described using various experimental approaches as flexible molecules which generate ensembles populating at least a small set of different conformations in solution. However, due to limitations of each approach in its ability to delineate the ensemble of conformations, an accurate and quantitative description of certain conformational features has not been performed for all DNMPs. Here, we apply a temperature replica-exchange molecular dynamics approach to fully and quickly converge conformational distributions of all RNA DNMPs immersed in the TIP3P water model using the AMBER ff14 force field. For a selection of DNMPs, the conformational ensembles were also generated when immersed in the OPC water model using alternative AMBER and CHARMM force fields. The OPC water model and other force field choices did not introduce new conformational classes but shifted the populations among existing conformations. Except for pyrimidine-pyrimidine dinucleosides, all other DNMPs populated four major conformations (which are defined in the main text and labeled A-form, Ladder, Inverted, and Sheared), in addition to an Extended form. Pyrimidine-pyrimidines did not generate the Sheared conformation. Distinguishing features and stabilizing factors of each conformation were identified and assessed based on the known experimental interpretations. The configuration of the glycosidic bond and the nonbonding interactions of hydrogen bond acceptors with the 2'-hydroxyl group were found to play determining roles in stabilizing particular conformations which could serve as a guide for potential force field modifications to improve the accuracy. Additionally, we computed stacking free energies based on the DNMP conformational distributions and found significant discrepancies with a previous study. Our investigation determined that the AMBER force field was incorrectly implemented in the previous study. In the future, this simulation approach can be used to quickly analyze the effects of new force field modifications in shifting the conformational populations of DNMPs,and can can be further applied to foresee such effects in larger RNA motifs including tetranucleotides and tetraloops.

Synthesis of Conformationally-Locked cis- and trans-Bicyclo[4.4.0] Mono-, Di-, and Trioxadecane Modifications of Galacto- and Glucopyranose; Experimental Limiting 3JH,H Coupling Constants for the Estimation of Carbohydrate Side Chain Populations and Beyond.

Hexopyranose side chains populate three staggered conformations, whose proportions can be determined from the three sets of ideal limiting 3JH5,H6R and 3JH5,H6S coupling constants in combination with the time-averaged experimental coupling constants. Literature values for the limiting coupling constants, obtained by the study of model compounds, the use of the Haasnoot-Altona and related equations, or quantum mechanical computations, can result in computed negative populations of one of the three ideal conformations. Such values arise from errors in the limiting coupling constants and/or from the population of nonideal conformers. We describe the synthesis and analysis of a series of cis- and trans-fused mono-, di-, and trioxabicyclo[4.4.0]octane-like compounds. Correction factors for the application of data from internal models (-CH(OR)-CH(OR)-) to terminal systems (-CH(OR)-CH2(OR)) are deduced from comparison of further models, and applied where necessary. Limiting coupling constants so-derived are applied to the side chain conformations of three model hexopyranosides, resulting in calculated conformer populations without negative values. Although, developed primarily for hexopyranose side chains, the limiting coupling constants are suitable, with the correction factors presented, for application to the side chains of higher carbon sugars and to conformation analysis of acyclic diols and their derivatives in a more general sense.

Función de los confórmeros de ataque cercano en la acilación enantioselectiva del (R,S)-propranolol catalizada por lipasa B de Candida antarctica

La lipasa B de Candida antarctica (CalB) se ha utilizado en la acilación quimio- y enantioselectiva del racemato (R,S)-propranolol. CalB tiene enantioselectividad moderada (E=63) por el R-propranolol. La enantioselectividad, se origina en la reacción de transferencia del grupo acilo desde la serina catalítica, acilada, al propranolol. La fase inicial de esta reacción involucra la formación de complejos de Michaelis y posteriormente conformaciones de ataque cercano. El análisis de las conformaciones de ataque cercano ha permitido en varios casos explicar el origen de la catálisis o reproducir el efecto catalítico. En este trabajo se profundiza en la comprensión la función de las conformaciones de ataque cercano en la enantioselectividad de la acilación del (R,S)-propranolol catalizada por CalB. Para lo anterior se realizó un estudio detallado de los complejos de Michaelis y de las conformaciones de ataque cercano del paso enantioselectivo de la reacción de acilación del (R,S)-propranolol utilizando un protocolo de dinámica molecular QM/MM (SCCDFTB/CHARMM) utilizando 6 distribuciones de velocidades iniciales y simulaciones de 2,5 ns. Se estudiaron 7 complejos CalB-propranolol. Los enlaces de hidrógeno del sitio activo de CalB acilada relevantes para la actividad catalítica fueron estables en todas las simulaciones. Las poblaciones de los complejos de Michaelis y de las conformaciones de ataque cercano son dependientes de la distribución de las velocidades iniciales de la dinámica molecular. La enantioselectividad moderada de CalB acilada, encontrada experimentalmente, puede ser parcialmente atribuida a la alta población de conformaciones de ataque cercano observada para el S-propranolol.