Rotamer Populations Research Articles

Earliest events in α-synuclein fibrillation probed with the fluorescence of intrinsic tyrosines

The fluorescence of the four tyrosines of α-synuclein (Syn) was used for probing the earliest events preceding the fibrillation of Syn, during the onset of the so-called lag-time of fibrillation. Steady-state fluorescence experiments revealed an increase in the fluorescence intensity (FI) for Syn solutions at pH values 3 and 2, in comparison with pH7, and fluorescence decays indicated that the FI increase did not result from suppression of excited-state proton transfer from the tyrosines to aspartates and glutamates, exposure of tyrosines to more hydrophobic environments, or reduction of homo-energy transfer. Instead, the FI increase was due to changes in the population of the tyrosine rotamers at low pH values.Stopped-flow experiments (pH-jumps) showed that the FI enhancement involves two processes: a fast (sub-7ms) intramolecular (concentration-independent) process, which we assign to the protein collapse at low pH, and a slower intermolecular (concentration-dependent) process of protein dimerization/oligomerization, starting at 4–10s after acidification.To the best of our knowledge, this is the first work on the experimental detection of these earliest processes in the fibrillation of Syn.

Population shuffling between ground and high energy excited states.

Stochastic processes powered by thermal energy lead to protein motions traversing time-scales from picoseconds to seconds. Fundamental to protein functionality is the utilization of these dynamics for tasks such as catalysis, folding, and allostery. A hierarchy of motion is hypothesized to connect and synergize fast and slow dynamics toward performing these essential activities. Population shuffling predicts a "top-down" temporal hierarchy, where slow time-scale conformational interconversion leads to a shuffling of the free energy landscape for fast time-scale events. Until now, population shuffling was only applied to interconverting ground states. Here, we extend the framework of population shuffling to be applicable for a system interconverting between low energy ground and high energy excited states, such as the SH3 domain mutants G48M and A39V/N53P/V55L from the Fyn tyrosine kinase, providing another tool for accessing the structural dynamics of high energy excited states. Our results indicate that the higher energy gauche - rotameric state for the leucine χ2 dihedral angle contributes significantly to the distribution of rotameric states in both the major and minor forms of the SH3 domain. These findings are corroborated with unrestrained molecular dynamics (MD) simulations on both the major and minor states of the SH3 domain demonstrating high correlations between experimental and back-calculated leucine χ2 rotameric populations. Taken together, we demonstrate how fast time-scale rotameric side-chain population distributions can be extracted from slow time-scale conformational exchange data further extending the scope and the applicability of the population shuffling model.

Temperature Dependence of Hydroxymethyl Group Rotamer Populations in Cellooligomers.

Empirical force fields for computer simulations of carbohydrates are often implicitly assumed to be valid also at temperatures different from room temperature for which they were optimized. Herein, the temperature dependence of the hydroxymethyl group rotamer populations in short oligosaccharides is investigated using molecular dynamics simulations and NMR spectroscopy. Two oligosaccharides, viz., methyl β-cellobioside and β-cellotetraose were simulated using three different carbohydrate force fields (CHARMM C35, GLYCAM06, and GROMOS 56Acarbo) in combination with different water models (SPC, SPC/E, and TIP3P) using replica exchange molecular dynamics simulations. For comparison, hydroxymethyl group rotamer populations were investigated for methyl β-cellobioside and cellopentaose based on measured NMR (3)JH5,H6 coupling constants, in the latter case by using a chemical shift selective NMR-filter. Molecular dynamics simulations in combination with NMR spectroscopy show that the temperature dependence of the hydroxymethyl rotamer population in these short cellooligomers, in the range 263-344 K, generally becomes exaggerated in simulations when compared to experimental data, but also that it is dependent on simulation conditions, and most notably properties of the water model.

CHARMM Drude Polarizable Force Field for Aldopentofuranoses and Methyl-aldopentofuranosides

An empirical all-atom CHARMM polarizable force filed for aldopentofuranoses and methyl-aldopentofuranosides based on the classical Drude oscillator is presented. A single electrostatic model is developed for eight different diastereoisomers of aldopentofuranoses by optimizing the existing electrostatic and bonded parameters as transferred from ethers, alcohols, and hexopyranoses to reproduce quantum mechanical (QM) dipole moments, furanose-water interaction energies and conformational energies. Optimization of selected electrostatic and dihedral parameters was performed to generate a model for methyl-aldopentofuranosides. Accuracy of the model was tested by reproducing experimental data for crystal intramolecular geometries and lattice unit cell parameters, aqueous phase densities, and ring pucker and exocyclic rotamer populations as obtained from NMR experiments. In most cases the model is found to reproduce both QM data and experimental observables in an excellent manner, whereas for the remainder the level of agreement is in the satisfactory regimen. In aqueous phase simulations the monosaccharides have significantly enhanced dipoles as compared to the gas phase. The final model from this study is transferrable for future studies on carbohydrates and can be used with the existing CHARMM Drude polarizable force field for biomolecules.

Microsecond Motion Modulates Ubiquitin Binding through an Allosteric Backbone/Side Chain Network

Motion is involved in a large number of protein functions. Relaxation dispersion (RD) NMR experiments sensitively probe microsecond to millisecond motions. We conducted an in-depth RD analysis of the backbone and side chain methyl groups of ubquitin. This survey showed a large number of atoms (>30) with microsecond fluctuations. These atoms are distributed throughout the structure. Strikingly, nearly all show the same exchange rate, which suggests that ubiquitin undergoes collective motion involving both the backbone and side chains. Furthermore, comparison of different methyl nuclei indicates that the nature of the side chain fluctuations is almost entirely due to changes in rotamer populations. Thus, collective microsecond backbone motion is coupled to redistribution of side chain rotamer populations through a mechanism we term “population shuffling”. We present a single collective mode of motion that yields a reaction coordinate corresponding to the relaxation dispersion data. The resulting model indicates that a localized conformational switch distant from the binding interface propagates changes throughout the structure. Analysis of crystal structures confirms this allosteric network and suggests that the microsecond motion modulates binding to particular interaction partners.

A Monte Carlo Method for Generating Side Chain Structural Ensembles

We report a Monte Carlo side chain entropy (MC-SCE) method that uses a physical energy function inclusive of long-range electrostatics and hydrophobic potential of mean force, coupled with both backbone variations and a backbone dependent side chain rotamer library, to describe protein conformational ensembles. Using the MC-SCE method in conjunction with backbone variability, we can reliably determine the side chain rotamer populations derived from both room temperature and cryogenically cooled X-ray crystallographic structures for CypA and H-Ras andNMR J-coupling constants for CypA, Eglin-C, and the DHFR product binary complexes E:THF and E:FOL. Furthermore, we obtain near perfect discrimination between a protein's native state ensemble and ensembles of misfolded structures for 55 different proteins, thereby generating far more competitive side chain packings for all of these proteins and their misfolded states.

A Combined Experimental and Theoretical Study of Conformational Preferences of Molecular Semiconductors

Structural modules used for assembling molecular semiconductors have typically been chosen to give desirable optical and electronic properties. Growing evidence shows that chemical functionalities should be considered for controlling molecular shape, which is important for function because of its influence on polymer secondary structure, lattice arrangements in crystals, and crystallization tendencies. Using density functional theory (DFT) calculations, followed by a natural bond orbital (NBO) analysis, we examine eight molecular semiconductors with resolved single crystal X-ray structures to understand the features that dominate molecular conformations and ultimately develop practical rules that govern these preferences. All molecules can be described by a D′–A–D–A–D′ architecture and have a 4,4-dimethyl-4H-silolo[3,2-b:4,5-b′]dithiophene (DTS) donor (D) core unit, with [1,2,5]thiadiazolo[3,4-c]pyridine (PT), 5-fluorobenzo[c][1,2,5]thiadiazole (FBT), or benzo[1,2,5]thiadiazole (BT) electron acceptor (A) units, and either thiophene, 5-hexyl-2,2′-bithiophene, or benzofuran electron-donating end-caps (D′). The NBO analysis shows that the energy difference between the two alternative conformations, or rotamers, (ΔErot) is a delicate balance of multiple competing nonbonding interactions that are distributed among many atoms. These interactions include attractive “donor–acceptor” electron sharing, steric repulsion, and electrostatic stabilization or destabilization. A proper grouping of these interactions reveals two primary factors determining ΔErot. The first concerns heteroatoms adjacent to the bonds connecting the structural units, wherein the asymmetric distribution of π-electron density across the link joining the units results in stabilization of one of two rotamers. The second factor arises from electrostatic interactions between close-contact atoms, which may also shift the ΔErot of the two rotamers. When all these constituent interactions cooperate, the dihedral angle is “locked” in a planar conformation with a negligible population of alternative rotamers.

Hidden regularity and universal classification of fast side chain motions in proteins.

Proteins display characteristic dynamical signatures that appear to be universal across all proteins regardless of topology and size. Here, we systematically characterize the universal features of fast side chain motions in proteins by examining the conformational energy surfaces of individual residues obtained using enhanced sampling molecular dynamics simulation (618 free energy surfaces obtained from 0.94 μs MD simulation). The side chain conformational free energy surfaces obtained using the adaptive biasing force (ABF) method for a set of eight proteins with different molecular weights and secondary structures are used to determine the methyl axial NMR order parameters (O(axis)(2)), populations of side chain rotamer states (ρ), conformational entropies (S(conf)), probability fluxes, and activation energies for side chain inter-rotameric transitions. The free energy barriers separating side chain rotamer states range from 0.3 to 12 kcal/mol in all proteins and follow a trimodal distribution with an intense peak at ~5 kcal/mol and two shoulders at ~3 and ~7.5 kcal/mol, indicating that some barriers are more favored than others by proteins to maintain a balance between their conformational stability and flexibility. The origin and the influences of the trimodal barrier distribution on the distribution of O(axis)(2) and the side chain conformational entropy are discussed. A hierarchical grading of rotamer states based on the conformational free energy barriers, entropy, and probability flux reveals three distinct classes of side chains in proteins. A unique nonlinear correlation is established between O(axis)(2) and the side chain rotamer populations (ρ). The apparent universality in O(axis)(2) versus ρ correlation, trimodal barrier distribution, and distinct characteristics of three classes of side chains observed among all proteins indicates a hidden regularity (or commonality) in the dynamical heterogeneity of fast side chain motions in proteins.

Conformational Properties of α- or β-(1→6)-Linked Oligosaccharides: Hamiltonian Replica Exchange MD Simulations and NMR Experiments

Conformational sampling for a set of 10 α- or β-(1→6)-linked oligosaccharides has been studied using explicit solvent Hamiltonian replica exchange (HREX) simulations and NMR spectroscopy techniques. Validation of the force field and simulation methodology is done by comparing calculated transglycosidic J coupling constants and proton–proton distances with the corresponding NMR data. Initial calculations showed poor agreement, for example, with >3 Hz deviation of the calculated 3J(H5,H6R) values from the experimental data, prompting optimization of the ω torsion angle parameters associated with (1→6)-linkages. The resulting force field is in overall good agreement (i.e., within ∼0.5 Hz deviation) from experimental 3J(H5,H6R) values, although some small limitations are evident. Detailed hydrogen bonding analysis indicates that most of the compounds lack direct intramolecular H-bonds between the two monosaccharides; however, minor sampling of the O6···HO2′ hydrogen bond is present in three compounds. The results verify the role of the gauche effect between O5 and O6 atoms in gluco- and manno-configured pyranosides causing the ω torsion angle to sample an equilibrium between the gt and gg rotamers. Conversely, galacto-configured pyranosides sample a population distribution in equilibrium between gt and tg rotamers, while the gg rotamer populations are minor. Water radial distribution functions suggest decreased accessibility to the O6 atom in the (1→6)-linkage as compared to the O6′ atom in the nonreducing sugar. The role of bridging water molecules between two sugar moieties on the distributions of ω torsion angles in oligosaccharides is also explored.

Side Chain Conformational Averaging in Human Dihydrofolate Reductase

The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of 3JCγCO and 3JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.

Understanding Side-Chain Motions using Statistical Torsion Angle Potential

To understand protein functions, an accurate protein structure is required. Because protein side-chain affects protein-protein interaction and protein-molecule interaction in tertiary protein structure, side-chain mobility is particularly important for understanding protein function. In general, determination of side-chain population is experimentally difficult, so we used computational method: Statistical Torsion Angle Potential (STAP) with Φ-χ1, Ψ-χ1 and χ1-χ2 combinations and simulated annealing protocol are used to predict side-chain mobility. Order parameters (S2) of methyl carbon and their correlation coefficients are evaluated for accuracy check. The computational prediction of χ1, χ2 rotamer population is performed for 17 target proteins. Seven target proteins are used for training set to find optimal values of van der Waals and STAP force constant. Ten target proteins are used for test set. This study can be used to characterize the χ1, χ2 conformation of exposed residues and to understand side-chain motions.

Effect of Short- and Long-Range Interactions on Trp Rotamer Populations Determined by Site-Directed Tryptophan Fluorescence of Tear Lipocalin

In the lipocalin family, the conserved interaction between the main α-helix and the β-strand H is an ideal model to study protein side chain dynamics. Site-directed tryptophan fluorescence (SDTF) has successfully elucidated tryptophan rotamers at positions along the main alpha helical segment of tear lipocalin (TL). The rotamers assigned by fluorescent lifetimes of Trp residues corroborate the restriction expected based on secondary structure. Steric conflict constrains Trp residues to two (t, g −) of three possible χ1 (t, g −, g +) canonical rotamers. In this study, investigation focused on the interplay between rotamers for a single amino acid position, Trp 130 on the α-helix and amino acids Val 113 and Leu 115 on the H strand, i.e. long range interactions. Trp130 was substituted for Phe by point mutation (F130W). Mutations at positions 113 and 115 with combinations of Gly, Ala, Phe residues alter the rotamer distribution of Trp130. Mutations, which do not distort local structure, retain two rotamers (two lifetimes) populated in varying proportions. Replacement of either long range partner with a small amino acid, V113A or L115A, eliminates the dominance of the t rotamer. However, a mutation that distorts local structure around Trp130 adds a third fluorescence lifetime component. The results indicate that the energetics of long-range interactions with Trp 130 further tune rotamer populations. Diminished interactions, evident in W130G113A115, result in about a 22% increase of α-helix content. The data support a hierarchic model of protein folding. Initially the secondary structure is formed by short-range interactions. TL has non-native α-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows α-helix to β-sheet transitions. The SDTF method is a valuable tool to assess long-range interaction energies through rotamer distribution as well as the characterization of low-populated rotameric states of functionally important excited protein states.

Allosteric Communication in the KIX Domain Proceeds through Dynamic Repacking of the Hydrophobic Core

The KIX domain of the transcriptional coactivator CREB binding protein (CBP) co-operatively mediates interactions between transcription factors. Binding of the transcription factor mixed-lineage leukemia (MLL) induces the formation of a low-populated conformer of KIX that resembles the conformation of the KIX domain in the presence of a second transcription factor molecule. NMR spin relaxation studies have previously shown that allosteric coupling proceeds through a network of hydrophobic core residues that bridge the two binding sites. Here we describe high-resolution NMR solution structures of the binary complex of KIX with MLL and the ternary complex of KIX formed with MLL and phosphorylated kinase inducible domain of CREB (pKID) as a second ligand. We show that binding of pKID to the binary complex of KIX with MLL is accompanied by a defined repacking of the allosteric network in the hydrophobic core of the protein. Rotamer populations derived from methyl group 13C chemical shifts reveal a dynamic contribution to the repacking process that is not captured by the structural coordinates and exemplify the dynamic nature of allosteric communication in the KIX domain.

Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle

Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of (3)JCγCO and (3)JCγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of (3)J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The (3)JCγCO and (3)JCγN coupling constants are found to be extremely sensitive measures of side chain χ1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.

Conformational properties of glucosyl-thioglucosides and their S-oxides in solution

A stereochemical study of a series of alkyl glucosyl-β-(1→6)-thioglucosides and their S-oxides by means of nuclear magnetic resonance and circular dichroism revealed that the populations around the thioglucosidic bond (ring I) as well as those of the interglycosidic linkage ω depend on the aglycone, the solvent and, in the S-oxides, on the absolute configuration of the sulfur atom. The results for the thio-disaccharides showed the strong influence of the solvent polarity on the conformational preferences of the interglycosidic bond. In polar solvents, the magnitudes of the rotamer populations, Pgg and Pgt, remained practically constant through the series, while in non-polar solvents a clear predominance of gt conformation was observed as well as the influence of the aglycone on the conformational equilibrium. The results for both (SS)- and (RS)-alkyl thiogentiobiosyl S-oxide series showed a clear predominance of the gt rotamer, Pgt always having a higher magnitude in the latter series than in the former. Both series exhibited linear correlations between interglycosidic Pgg and Pgt and Taft’s steric parameter (ES) for the alkyl group attached to the sulfinyl group, especially in non-polar solvents. The stereochemical study around the C1–S bond established that the flexibility around this linkage depends on aglycone size, solvent polarity, and the absolute configuration of the sulfur, derivatives of the SS series showing higher flexibility in both polar and non-polar media. The conformational properties of these compounds in solution are explained in terms of stereoelectronic, steric, and solvent effects.

Structural Refinement from Restrained-Ensemble Simulations Based on EPR/DEER Data: Application to T4 Lysozyme

DEER (double electron-electron resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parametrized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement.

Molecular Recognition through Concerted Ubiquitin Backbone and Side Chain Motion Determined from NMR and MD Simulations

Motion is involved in a large number of protein functions. For ubiquitin, residual dipolar coupling (RDC) derived ensembles have suggested it recognizes binding partners via conformational selection through motions occurring on the supra tau-c (>4 ns) timescale [1]. Subsequent relaxation dispersion (RD) studies identified microsecond fluctuations for six backbone atoms [2,3]. However, it has not been clear if these motions are independent or collective, and what role side chains play. To address those questions, we have conducted an in-depth RD analysis of the backbone and side chain methyl groups. This survey showed a large number of atoms (>30) with microsecond fluctuations. These atoms are distributed throughout the structure and nearly all show the same exchange rate, which suggests that ubiquitin undergoes collective motion involving both the backbone and side chains. The presence of microsecond side chain motion agrees with previous RDC measurements of methyl groups within ubiquitin [4]. Furthermore, comparison of different methyl nuclei indicates that the nature of the side chain fluctuations is almost entirely due to changes in rotamer populations. This is borne out in molecular dynamics simulations of ubiquitin trapped in different conformational states by binding partners [5]. Residues showing RD were significantly more likely to show changes in rotamer populations in the simulations. Put together, this leads to a model where collective microsecond backbone motion is coupled to redistribution of side chain rotamer populations. The correlation with binding partner simulations implicates this motion in conformational selection.1. Lange et al. Science. 320:1471-1475 (2008)2. Ban et al. Angew Chem Int Ed. 50:11437-1144 (2011)3. Ban et al. J Magn Reson. 221:1-4 (2012)4. Fares et al. J Biomol NMR. 45:23-44 (2009)5. Peters et al. PLoS Comput Biol. Accepted (2012)

Rotamer Library of Spin Labeled Cysteines Attached to T4 Lysozyme Deduced from Molecular Dynamics Simulations Constrained by Double Electron-Electron Resonance (Deer) Experiments

DEER (Double Electron Electron Resonance) is a powerful pulsed EPR technique allowing the determination of spin-spin distance histograms between side-directed nitroxide label sites on a protein in their native environment. In this study, a novel molecular dynamics simulation method, the restrained ensemble (RE) method, has been used for the refinement of spin labels inserted in T4 Lysozyme (T4L) structures. In the RE method, the spin-spin distance distribution histograms calculated from a multiple-copy molecular dynamics simulations are forced, via a special ensemble-based energy restraint, to match those extracted from 51 EPR/DEER experimental spin-spin pairs. To examine the effect of the restraint on the rotameric state of the spin label at the 37 different sites in T4L, conventional long molecular dynamics (MD) and locally enhanced sampling (LES) simulations were also performed. As expected, the distance histograms obtained from the RE simulations are very similar to those obtained from experiment; however, the distance histograms from conventional long MD and LES simulations deviate from experiment. From the analysis of these simulations, a general trend is found in rotamer population distribution along five dihedral angles connecting the nitroxide ring to the protein backbone. Overall, the rotamer population obtained from the RE simulation agrees with those obtained from x-ray structure. From the analysis of the dynamics of nitroxide atoms in spin label side chains, a force field for a pseudo nitroxide dummy atom has been parameterized. This pseudo nitroxide was used in an RE simulation coupled with the EPR/DEER experimental distance distribution data to refine distorted structures of T4L. Thus, experimental restraints from DEER experiments in conjunction with RE simulations can be used to refine structural propertied of biological systems.

Direct Synthesis of Maradolipids and Other Trehalose 6-Monoesters and 6,6′-Diesters

It was shown that reaction of trehalose with 1 equiv of a fatty acid in pyridine promoted by 1 equiv of the uronium-based coupling agent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) at room temperature gives a good yield of the primary ester accompanied by small amounts of the diprimary ester using hexanoic, palmitic, and oleic acids as examples. Reactions using 2 equiv of the fatty acids gave the symmetrical diesters. The monoesters were reacted with different fatty acids to give nonsymmetric 6,6'-diesters in very good yields. Compounds synthesized include the most abundant component of the very complex maradolipid mixture, 6-O-(13-methyltetradecanoyl)-6'-O-oleoyltrehalose, and a component potentially present in this mixture, 6-O-(12-methyltetradecanoyl)-6'-O-oleoyltrehalose, a derivative of an ante fatty acid. The C5-C6 rotameric populations of 6-O-monoesters, symmetrical 6,6'-diesters, and 2,6,6'-triesters of fatty acids were calculated from the values of the H5-H6R and H5-H6S coupling constants and found to be similar to those found for glucose. The rotameric populations of the monosubstituted glucose residues in the 2,6,6'-triesters was altered considerably to favor the gt rotamer, presumably because of attraction between the 2- and 6'-fatty acid chains.

Performance of the SCC-DFTB Model for Description of Five-Membered Ring Carbohydrate Conformations: Comparison to Force Fields, High-Level Electronic Structure Methods, and Experiment.

The solution conformations of biologically important mono- and di-α-d-arabinofuranosides were investigated using the dispersion-corrected self-consistent charge density functional tight binding (SCC-DFTB) and the AMBER/GLYCAM models. Simulations were performed using both long dynamics and umbrella sampling simulations. Angular distributions about the exocyclic C-C bonds and puckering distributions of the rings obtained from the SCC-DFTB model were quite different from those obtained with the AMBER/GLYCAM approach. The joint probability distribution of rotamer and ring puckering parameters reveals further discrepancies, while both methods predict weak correlation between exocyclic torsions and ring puckering. To assess the reliability of the simulations, ensemble-averaged vicinal proton-proton coupling constants ((3)JH,H) were calculated and compared directly to experimental NMR coupling constants. It is found that the (3)JH,H values obtained from the AMBER/GLYCAM simulations agree with experiment, while those obtained from the SCC-DFTB method, in most cases, differ from experimental (3)JH,H values. Potential energy surfaces (PES) along the exocyclic torsion obtained from ab initio and DFT calculations differ significantly from those obtained with the SCC-DFTB method. This study establishes that a high-quality all-atom force field would be more suitable than one of the superior semiempirical methods, SCC-DFTB, for investigation of rotamer populations and ring puckering in floppy ring systems such as furanosides.