Higher Energy Conformers Research Articles

揺らぐ蛋白質構造の解析 ―可変圧力ＮＭＲからのアプローチ

Protein structure in solution fluctuates over a wide range of conformation, but the reality of the fluctuation has been little explored. In this article, we present an approach to this problem using variable pressure NMR spectroscopy. The method enables us to explore protein conformation over a wide range of conformational space than hitherto explored by conventional NMR method and brings about a new dynamic view on protein structure. The highlight of the method is the determination of the structural coordinates of semi-stable higher energy conformers that exist in low population in dynamic equilibrium with the stable native conformer under physiological conditions. Available evidence suggests that such high energy conformers play crucial roles in function in many proteins.

Contortions of encapsulated alkyl groups

Rotors are recalled as early molecular devices that transmit information through changes in conformation. Specific cases involve bipyridyls and biphenyls in which the biaryl bond acts as a fulcrum to relay applied stresses from one site to another. New types of molecular stress encountered by encapsulated molecules are identified--including bending, straightening, squeezing, grinding and compression. For flexible molecules in reversibly formed capsules a fluid model of recognition is proposed that is neither lock-and-key nor induced fit. Instead, the guest assumes the shape that best fills the available space, even if contortions to higher energy conformations are required. For encapsulated alkanes, a delicate balance of attraction and repulsion exists when the size of a guest molecule approaches the space available to it. The complexes are analyzed by both NMR and computational methods and detailed maps of the host-guest interfaces in solution are provided. The reversible transition of an encapsulated alkane between a compressed, coiled conformation and a relaxed, extended one is described. The system is a spring-loaded molecular device under the control of acids and bases that offers an alternative to the rotors of current molecular machinery.

UV Photolysis Products of Propiolic Acid in Noble-Gas Solids

Photolysis (193 nm) of propiolic acid (HCCCOOH) was studied with Fourier transform infrared spectroscopy in noble-gas (Ar, Kr, and Xe) solid matrixes. The photolysis products were assigned using ab initio quantum chemistry calculations. The novel higher-energy conformer of propiolic acid was efficiently formed upon UV irradiation, and it decayed back to the ground-state conformer on a time scale of approximately 10 min by tunneling of the hydrogen atom through the torsional energy barrier. In addition, the photolysis produced a number of matrix-isolated 1:1 molecular complexes such as HCCH...CO2, HCCOH...CO, and H2O...C3O. The HCCH...CO2 complex dominated among the photolysis products, and the computations suggested a parallel geometry of this complex characterized by an interaction energy of -9.6 kJ/mol. The HCCOH...CO complex also formed efficiently, but its concentration was strongly limited by its light-induced decomposition. In this complex, the most probable geometry was found to feature the interaction of carbon monoxide with the OH group via the carbon atom, and the computational interaction energy was determined to be -18.3 kJ/mol. The formation of the strong H2O...C3O complex (interaction energy -21 kJ/mol) was less efficient, which might be due to the inefficiency of the involved radical reaction.

S,S-1,2-Dicyclohexylethane-1,2-diol and its racemic compound: a striking exception to Wallach's rule

The structures of enantiopure S,S-1,2-dicyclohexylethane-1,2-diol and its racemic compound (rac-S,S-1,2-dicyclohexylethane-1,2-diol) have been determined at 295 and 173 K. The crystals of the enantiopure material are more than 4% denser than the crystals of the racemic compound, but the melting points indicate that the crystals of the less dense racemic compound are considerably more stable than those of the racemic conglomerate. This apparent exception to the correlation of crystal density and melting point is explained. The enantiopure crystals have four molecules in the asymmetric unit (Z' = 4). Two of the molecules have the conformation observed for the one independent molecule of the racemic compound and two have a higher energy conformation; the overall P2(1) structure is a perturbed version of a P2(1)2(1)2(1) structure with Z' = 2. The enantiopure and racemic crystals have the same hydrogen-bonding motif, but the motif in the former appears to be significantly strained. A reason why crystals of enantiopure material might be systematically less dense than crystals of its racemic compound and to be more likely to have Z' > 1 is suggested.

Spectroscopic and theoretical studies of some p-substituted α-methylthio-α-diethoxyphosphorylacetophenones

The analysis of the IR carbonyl band of the α-methylthio-α-diethoxyphosphoryl p-substituted acetophenones p-Y-Ph-C(O)CH[SMe][P(O)(OEt) 2] (Y = OMe 1, H 2, F 3, Cl 4, Br 5 and NO 2 6), supported by HF/6-31G** ab initio calculations of the α-methylthio-α-diethoxyphosphoryl acetophenone 2, indicated the existence of a single stable c 1 conformer in gas phase and in solvents of increasing polarity, along with the presence of a second high energy conformation in gas phase. The c 1 conformer presents the (SMe) group in a syn-clinal ( gauche) geometry and the [P(O)(OEt 2)] group in a quasi-periplanar ( quasi-cis) geometry with respect to the carbonyl group, which is stabilized by the synergism of the n S/π* CO and σ C–S/ π* CO orbital interactions and O δ − ( PO ) ⋯ C δ + ( CO ) orbital and Coulombic interactions, acting to increase the carbonyl oxygen negative charge, and in turn facilitates the O δ − ( CO ) ⋯ P δ + ( PO ) electrostatic interaction. The negative and almost constant carbonyl frequency shifts (Δ ν) of ca. −11 cm −1 for compounds 1– 6 relative to the parent acetophenones 7– 12, in CCl 4, corroborate the prevalence of the electronic interactions over the (− I σ) inductive effect of the α-substituents for the title compounds. The X-ray diffraction analysis for 3 indicates that it exists in the solid state in the c 1 ′ conformation, which is stabilized by the intramolecular O δ− (1) [CO}⋯ P δ+ (5) [PO] and H δ+ (10) [SMe]⋯O δ− (6) [PO] orbital and electrostatic (hydrogen bond) interactions. Moreover, these molecules form dimers which are stabilized through intermolecular hydrogen bonds H δ+ (4) [CH]⋯O δ− (1) [CO], H δ+ (5′) [Ph]⋯ O δ− (6) [PO] and H δ+ (6′) [Ph]⋯ O δ− (6) [PO].

Ab initio DFT calculations of the configurational and conformational preferences of 2-phenylsulfinylcyclohexanones — Evidence for "exo-anomeric" interactions

The conformational analysis of 2-phenylsulfinylcyclohexanone by ab initio density functional calculations is described. Six conformations corresponding to axial/equatorial isomers and rotation about the exocyclic C2—S bond in each of the RR or RS diastereomers were calculated and the results were examined in terms of relative energies, electrostatic interactions, orbital interactions, and geometrical variations. The global minimum conformation was the RS isomer that positioned the phenylsulfinyl moiety in an equatorial orientation and the sulfinyl oxygen in an anti orientation with respect to the carbonyl carbon atom. Of the other three low energy conformations, only one had a gauche arrangement of these atoms, and only in one of the four lower energy conformations was evidence found for a S-O(–)···C(+)-O electrostatic interaction. In contrast, the results were consistent with the operation of nS → [Formula: see text] stabilizing orbital interactions. Further support for this hypothesis was obtained from the increased C=O bond lengths in these four conformations relative to the other conformations, and by the torsional angle distortion away from ideal geometry, presumably to maximize the stabilizing orbital interaction. We propose that this conformational preference is a manifestation of a generalized exo-anomeric effect. The longer C2—S bond in the axial isomers was also interpreted in terms of a stabilizing πC=O → [Formula: see text] interaction, analogous to an endo-anomeric interaction. Comparison of the computational results to available experimental data on the conformational equilibrium of each diastereomer in solution suggests which conformers are present in each of the equilibria. The available data for the solid state indicate that the RR and RS isomers both crystallize in high energy conformations, stabilized by intermolecular interactions.Key words: 2-phenylsulfinylcyclohexanones, configurational isomers, conformational preferences, DFT calculations, exo-anomeric effect.

Molecular dynamics simulations of a cyclic-β-(1→2) glucan containing an α-(1→6) linkage as a ‘molecular alleviator’ for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via β-(1→2) linkages excluding one α-(1→6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All β-(1→2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the α-(1→6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only β-(1→2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The α-(1→6)-glycosidic linkage can relieve this constraint more efficiently than the β-(1→2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the α-(1→6)-glycosidic linkage.

Hula-twist photoisomerization of a styrene derivative examined by argon-matrix-infrared-spectroscopic method

IR spectra of α-cyano-o-fluorocinnamonitrile, its vinyl deuterated analogue and the corresponding photoproducts were recorded under Ar matrix isolation conditions (13 K). An analysis of the IR spectra, assisted by calculations of the expected absorption signals from these structures, showed that the photoproducts were consistent with the previously assigned Hula-twist (HT) product, an unstable high energy conformer of the parent system. The absence of a significant deuterium isotope effect on rates of photochemical reaction appears to rule out the involvement of a hydrogen-tunnelling mechanism for the HT process.

Matrix isolation infrared and ab initio studies of the conformations of dimethoxymethylsilane

Conformers of dimethoxymethylsilane were studied using matrix isolation infrared spectroscopy, by trapping it in nitrogen and argon matrixes. In addition to a room temperature effusive source, supersonic and hot nozzle effusive sources were also used to deposit the matrix. These experiments were done to aid assigning the vibrational features, by altering the population of the various conformers in the matrix. As a result of these experiments, the infrared spectral features have been assigned to the ground state conformer, G ±G ∓ and higher energy conformers, G −G − and G −T. Our experimental observations were supported by computations performed at HF and B3LYP levels using 6-31++G** basis set.

Conformational Distinguishability of Medium Cycloalkanes in Crystals via Inelastic Neutron Scattering

This paper begins an exploration of the use of the combination of DFT computations with experimental inelastic neutron scattering (INS) spectra as a method for establishing what conformation is present in a molecular crystal at low temperature. Presented here are INS spectra of a series of medium-sized cycloalkanes: C6H12, C7H14, C8H16, C10H20, C12H24, and C14H28. Optimized geometries and normal mode calculations were performed at B3LYP/6-311G(d,p) on the lowest energy conformations (i.e., those thermally accessible at the experimental temperature of 30 K). The calculated and observed spectra were analyzed for the best fit from each set of conformers, allowing a prediction of the dominant conformation in a conformationally rich system. For each cycloalkane, the calculated spectrum for the lowest energy conformer shows good agreement with experiment while the higher energy conformations have a much poorer fit. With little ambiguity, the lowest energy conformer is therefore predicted to be the dominant conformation, consistent with the diffraction data available for C6H12, C10H20, C12H24, and C14H28. These results indicate that INS spectroscopy may be a useful tool in determining the dominant conformation in a crystal lattice in cases such as this in which the intermolecular interactions are weak and the different conformers are calculated to have distinguishable spectra. Such an analysis is applied to the cases of C7H14 and C8H16 for which no low-temperature X-ray analysis is available. Clear structure predictions result, and the conformer observed is that computed to be of lowest energy for the molecule in isolation.

Role of each residue in catalysis in the active site of pyrimidine nucleoside phosphorylase from Bacillus subtilis: A hybrid QM/MM study

Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. We have built a model of a closed active conformation of the three-dimensional structure of PYNP from Bacillus subtilis. Using docking, molecular dynamics, and hybrid quantum-mechanical/molecular-mechanical methods to study the reaction mechanics between PYNP and a substrate, we identified the role of each residue in the active site during the entire catalytic process. The results indicate that the function of His 82, Arg 169, and Lys 188 is to stabilize the uridine in a high-energy conformation by means of electrostatic interactions and that these residues are involved in catalysis. In addition, the function of Asp 162 is likely to activate Lys 188 for phosphorolytic catalysis through polarization effects.

可変圧力ＮＭＲ法による新しい蛋白質構造パラダイムの構築

A protein in solution is a thermodynamic entity existing in equilibria among multiple conformations from the folded to the unfolded. Various experimental results indicate that transitions to higher energy conformers are crucial for function in many proteins. However, our knowledge on higher energy conformers is extremely limited at present, compared to the knowledge about the folded counterparts. A newly emerging technique, the variable pressure NMR method, enables us to explore a much wider conformational space of proteins than hitherto explored by X-ray crystallography and conventional NMR technique, giving a new dynamic view of proteins that amply fluctuate over the entire conformational space.

Structures of Tryptophanyl-tRNA Synthetase II from Deinococcus radiodurans Bound to ATP and Tryptophan: INSIGHT INTO SUBUNIT COOPERATIVITY AND DOMAIN MOTIONS LINKED TO CATALYSIS

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.

Photochemistry of Salicylideneaniline Analogue at Low Temperature

Abstract Naphthalene-based salicylideneaniline analogue 2 underwent photochemical cis–trans isomerization in organic glass at low temperature. The lifetime of the photoproduct trans-keto tautomer in glass was similar to that in solution at higher temperature, suggesting the formation of high energy conformer by volume conserving isomerization mechanism.

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Lewis Acid Properties of Zinc(II) in Its Cyclen Complex. The Structure of [Zn(Cyclen)(SC(NH2)2](ClO4)2 and the Bonding of Thiourea to Metal Ions. Some Implications for Zinc Metalloenzymes

The structure of the complex [Zn(cyclen)Tu](NO(3))(2) (1) is reported (cyclen = 1,4,7,10-tetraazacyclododecane; Tu = thiourea): orthorhombic, space group P2(1)2(1)2(1), a = 11.4170(11) A, b = 12.1995(11) A, c = 12.5299(12) A, Z = 4, R = 0.0504. The coordination of the cyclen is the same as that found for other similar Zn(II) complexes, with square pyramidal coordination around the Zn(II) and mean Zn-N bond lengths of 2.16 A. The coordinated Tu occupies the axial coordination site, with Zn-S = 2.31 A. The Zn-S-C-N torsion angle, involving the coordinated Tu, of 75.4 degrees is unusually large, because such torsion angles involving coordinated Tu are normally closer to 0 degrees. The bonding between Zn and S is discussed in terms of overlap with the p orbitals on S, which favors the eclipsed (Zn-S-C-N torsion = 0 degrees) mode of coordination of Tu. The energies of eclipsed and staggered modes (Zn-S-C-N = 90 degrees) of coordination of Tu to metal ions are examined by means of ab initio calculations, using the STO-3G basis set. It is concluded that the rather low formation constant for the Tu complex with Zn(II)/cyclen reported in this work was due to steric effects in 1, which prevent the adoption of the lower energy eclipsed conformation. These steric effects, because of clashes that would occur between Tu in the eclipsed conformation and the cyclen ring, cause the coordination of Tu with a higher energy conformation, with Zn-S-C-N = 75.4 degrees. The latter approaches the high energy staggered conformation that has Zn-S-C-N = 90 degrees. log K(1) values for Cl(-), Br(-), I(-), and CN(-) are reported and shown to be consistent with the binding site on the Zn(II) in the Zn(II)/cyclen complex being softer in the hard and soft acids and bases (HSAB, Pearson 1997) sense than the Zn(II) aqua ion, but not as soft as Zn(II) in triaza macrocycles that promote tetrahedral coordination. The change in HSAB character from intermediate in the Zn(II) aqua ion to softer in the cyclen complex, and softer still in tridentate N-donor ligands in model complexes, and in the Zn(II) active site of carbonic anhydrase as representative of Zn(II) metalloenzymes in general, is discussed in terms of the role of such effects in the functioning of metalloenzymes.

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

A series of cyclic, disulfide- or dithioether-containing tetrapeptides based on previously reported potent mu- and delta-selective analogs has been explored with the aim of improving their poor affinity to the kappa-opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe(3) replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo-Arg), or aromatic sides chains (Trp, Tyr, p-NH(2)Phe), and with d-Pen(4) replaced by d-Cys(4), and binding affinities to stably expressed mu-, delta-, and kappa-receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the kappa-receptor, with the exception of the tetrapeptide Tyr-c[d-Cys-Phe-d-Cys]-NH(2), cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki(mu) = 1.26 nm, Ki(delta) = 16.1 nm, Ki(kappa) = 38.7 nm). Modeling of the kappa-receptor/ligand complex in the active state reveals that the receptor-binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the mu-receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d-Pen or d-Cys) and correlates well with the observed kappa-receptor binding affinity.

Comparative Analysis of Protein-Bound Ligand Conformations with Respect to Catalyst's Conformational Space Subsampling Algorithms

We examined the quality of Catalyst's conformational model generation algorithm via a large scale study based on the crystal structures of a sample of 510 pharmaceutically relevant protein-ligand complexes extracted from the Protein Data Bank (PDB). Our results show that the tested algorithms implemented within Catalyst are able to produce high quality conformers, which in most of the cases are well suited for in silico drug research. Catalyst-specific settings were analyzed, such as the method used for the conformational model generation (FAST vs BEST) and the maximum number of generated conformers. By setting these options for higher fitting quality, the average RMS values describing the similarity of experimental and simulated conformers were improved from an RMS of 1.06 with max. 50 FAST generated conformers to an RMS of 0.93 with max. 255 BEST generated conformers, which represents an improvement by 12%. Each method provides best fitting conformers with an RMS value<1.50 in more than 80% of all cases. We analyzed the computing time/quality ratio of various conformational model generation settings and examined ligands in high energy conformations. Furthermore, properties of the same ligands in various proteins were investigated, and the fitting qualities of experimental conformations from the PDB and the Cambridge Structural Database (CSD) were compared. One of the most important conclusions of former studies, the fact that bioactive conformers often have energy high above that of global minima, was confirmed.

Hydrogen exchange and ligand binding: Ligand‐dependent and ligand‐independent protection in the Src SH3 domain

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.

Ethyl anion preferred conformation

AbstractAb initio architecture and torsional barrier for ethyl anion, C2H are analyzed using natural bond orbital methodology. The B3LYP/6‐311++G(3df,2p) 2‐kcal/mol torsional barrier between the preferred staggered and higher‐energy eclipsed conformer is calculated to be one‐third lower than for ethane. This decrease is largely attributed to reduced hyperconjugative stabilization of the equilibrium anion conformer compared to that for ethane. Proton removal is also predicted to cause opening of the ethane central CCH angle by 7°, attributed to decreased steric repulsion and to increased hyperconjugative stabilization accompanying angle widening in the ion. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005