High Conformational Flexibility Research Articles

NMR study of hydroxy protons of di‐ and trimannosides, substructures of Man‐9

The chemical shifts, temperature coefficients and inter-residual rotating-frame Overhauser effect (ROE)s for the hydroxy protons of some alpha-(1,2)-, alpha-(1,3)- and alpha-(1,6)-linked di- and trimannosides have been measured for samples in 85% H2O/15% acetone-d6 solution. These mannosides, Manalpha(1-->2)ManalphaOMe (1) Manalpha(1-->3)ManalphaOMe (2), Manalpha(1-->6)ManalphaOMe (3), Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe (4), Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe (5), Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe (6) and Manalpha(1-->3)[Manalpha1-->6]ManalphaOMe (7), are substructures of the N-glycan Man-9. The NMR data show that the hydration of each individual hydroxyl group in the di- and trisaccharides is very similar to the hydration of the corresponding hydroxyl in the monomeric methyl alpha-D-mannoside. No hydrogen-bond interactions were found to stabilize the conformations of the alpha-(1,2)- and alpha-(1,6)-linkages and the chemical shifts for the hydroxy proton resonances of the alpha-(1,6)-linkage indicated high-conformational flexibility. For the alpha-(1,3)-linkage, however, the downfield shift for the signal of O(2)H of the 3-substituted residue together with the ROE between this proton and H5' on the next residue suggest some weak inter-residue interactions.

Evidence for the nucleosome-disruption process regulated by phosphorylation of 120 kDa protein complex in Drosophila embryo cell-free system

Using cell-free system derived from Drosophila embryos, we found evidence for a regulated nucleosome disruption process, which depends on the phosphorylation status of 120 kDa protein (complex). Dephosphorylation enables the remodeling activity to destabilize nucleosomes, which assume a more accessible structure, possessing increased DNase I sensitivity and high conformational flexibility of DNA; remodeling was more efficient on highly acetylated chromatin templates. This phosphorylation-regulated nucleosome destabilization, acting synergistically with histone acetylation, is discussed as a possible mechanism to provide regulated disrupt of histone-DNA interaction.

Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

Phospholemman (PLM) is a 72-residue bitopic cardiac transmembrane protein, which acts as a modulator of the Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger and possibly forms taurine channels in nonheart tissue. This work presents a high resolution structural model obtained from a combination of site-specific infrared spectroscopy and experimentally constrained high throughput molecular dynamics (MD) simulations. Altogether, 37 experimental constraints, including nine long range orientational constraints, have been used during MD simulations in an explicit lipid bilayer/water system. The resulting tetrameric alpha-helical bundle has an average helix tilt of 7.3 degrees and a crossing angle close to 0 degrees . It does not reveal a hydrophilic pore, but instead strong interactions between various residues occlude any pore. The helix-helix packing is unusual, with Gly(19) and Gly(20) pointing to the outside of the helical bundle, facilitating potential interaction with other transmembrane proteins, thus providing a structural basis for the modulatory effect of PLM on the Na(+)/K(+)-ATPase. A two-stage model of interaction between PLM and the Na(+)/K(+)-ATPase is discussed involving PLM-ATPase interaction and subsequent formation of an unstable PLM trimer, which readily interacts with surrounding ATPase molecules. Further unconstrained MD simulations identified other packing models of PLM, one of which could potentially undergo a conformational transition to an open pore.

Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin

The T-loop motif is an important recurrent RNA structural building block consisting of a U-turn sub-motif and a UA trans Watson–Crick/Hoogsteen base pair. In the presence of a hairpin stem, the UA non-canonical base pair becomes part of the UA-handle motif. To probe the hierarchical organization and energy landscape of the T-loop, we performed replica exchange molecular dynamics (REMD) simulations of the T-loop in isolation and as part of a hairpin. Our simulations reveal that the isolated T-loop adopts coil conformers stabilized by base stacking. The T-loop hairpin shows a highly rugged energy landscape featuring multiple local minima with a transition state for folding consisting of partially zipped states. The U-turn displays a high conformational flexibility both when the T-loop is in isolation and as part of a hairpin. On the other hand, the stability of the UA non-canonical base pair is enhanced in the presence of the UA-handle. This motif is apparently a key component for stabilizing the T-loop, while the U-turn is mostly involved in long-range interaction. Our results suggest that the stability and folding of small RNA motifs are highly dependent on local context.

Binding modes of protegrin-1, a beta-strand antimicrobial peptide, in lipid bilayers

Molecular dynamics (MD) simulations of the interactions of a beta-strand antimicrobial peptide, protegrin-1, with model lipid bilayers of different hydrophobic widths show that protegrin-1 possesses at least two distinct binding modes in the trans-membrane orientation. In one of the modes, three of the cationic arginine residues bind to one bilayer leaflet and the other three arginine residues bind to the opposite leaflet, while in the second binding mode, four arginines bind to one leaflet and two to the other. The existence of multiple binding modes is facilitated by the high conformational flexibility of the first four residues of the N-terminal region, with a sequence of RGGR. One of the binding modes causes a larger bilayer disruption than the other, and in either mode, the bilayer disruption is smaller for lipid membranes with smaller bilayer widths. Simulations of the self-assembly of initially random lipid/water mixtures in the presence of PG-1 peptides show that the system evolves into a bilayer, with a stable water pore spanning the two lipid leaflets. Lipid head groups and the PG-1 peptide stabilize the water pore.

Density Functional Theory Study of Triphenyl Phosphite: Molecular Flexibility and Weak Intermolecular Hydrogen Bonding

The high conformational flexibility of triphenyl phosphite (TPP) is investigated by density functional theory (DFT) calculations. First, through a scan of the molecular potential energy surface, we bring to light a new stable conformation of an isolated molecule, not yet encountered in the crystal states of TPP. Different relevant conformations of the TPP monomer in the gas state are further presented and discussed in terms of molecular structure, relative energy, and dipole moments. Second, we considered dimer and trimer of TPP starting from their structural topology within the hexagonal crystal, which is characterized by the existence of molecular rods. It is shown that weak C-H...O intermolecular hydrogen bonds in TPP cannot definitely be excluded, and finally this point is discussed in the scope of the glacial state problem.

Structural Preferences of Neuroprotective S14G-Humanin Peptide Analyzed by Molecular Modeling and Circular Dichroism

S14G-humanin (S14G-HN) is one of the latest of a new family of neuropeptides with protective action against Alzheimer's disease insults. The structure of S14G-HN was studied with both spectroscopic techniques and molecular dynamics simulation. Secondary structure predictions and modeling of backbone conformation were carried out. Side chain reconstruction, homology modeling and molecular dynamics (MD) simulations were performed on four different models. A beta strand tendency in residues 5 to 10 and a propensity to adopt turn or irregular conformation in residues 13 to 17 was found. Circular dichroism experimental studies of S14G-HN in aquaeous solution and in different 2,2,2-trifluoroethanol (TFE) concentrations were also performed. In the absence of TFE and at low TFE concentrations, CD spectra are indicative of a small degree of ordering in the peptide. On further increment of TFE concentration, changes occur that indicate the formation of a structured conformation. Both experimental and computational results indicate that S14G-HN has a reduced helical propensity, in contrast with wild type humanin, as well as a higher conformational flexibility.

Vesicles from docosahexaenoic acid

In dilute aqueous solution and at room temperature, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) self-assembles into vesicles (self-closed bilayers), if the molar ratio of the neutral form of DHA to anionic DHA is kept between 1:1 and 1:3 (corresponding to a bulk pH between 8.5 and 9.2 for a system with 10 mM DHA). By using polycarbonate membrane extrusion, stable unilamellar DHA vesicles with an average diameter of 80 nm can be prepared at pH 8.8. Cryo-transmission electron microscopy indicates that the width of the DHA bilayers in the vesicles is clearly below twice the length of an extended DHA molecule, indicating a high conformational flexibility of DHA within the vesicle bilayer. These DHA bilayers have a similar thickness like bilayers of vesicles prepared at pH 8.5 from oleic acid ( cis-9-octadecenoic acid). Using calcein as fluorescent reference compound, it is shown that water-soluble molecules can be encapsulated inside DHA vesicles which may make them interesting for medical or food applications.

Attractive halogen–halogen interactions: F 3CCl⋯FH and F 3CCl⋯FCH 3 dimers

The F 3CCl⋯FH and F 3CCl⋯FCH 3 dimers, which feature the halogen–halogen contacts, are investigated at MP2/6–311++G(d,p) and MP2/aug–cc–pVDZ levels of approximation. The binding energies of these complexes are found to be comparable to those of the weak hydrogen bonds. In both complexes the Cl⋯F are found to be significantly shorter than the sum of the corresponding van der Waals radii. The C–Cl⋯F contacts are also found to exhibit certain deviation from linearity. However, the energy differences between linear and bent structures are very small and primarily accounted for by electrostatic interactions between remote parts of the dimer. This indicates a high conformational flexibility of the halogen–halogen contacts and may help to explain the diversity of structural features in crystals formed by halogen-containing molecules. In both dimers the halogen–halogen interaction leads to certain shortening of the C–Cl electron accepting bond. This is accompanied by a small increase of the C–Cl stretching frequency. Hence, the two investigated dimers can possibly be classified as the blue-shifting halogen–halogen contacts.

Conformational studies on parathion

AbstractTheoretical conformational studies have been carried out at the density functional theory (DFT) level on parathion (O,O‐diethyl‐O‐p‐nitrophenyl phosphorothioate), to correlate the conformational properties of this molecule with its biological activity. Different conformers of parathion have been taken into account, and the accuracy of our theoretical approach has been gauged against the results of the second‐order Møller–Plesset perturbation calculations. The aqueous solvation of these molecules has been studied at the DFT level, using a polarized continuum model (PCM) with a conductor‐like screening reaction field approach. The results show that parathion has high conformational flexibility in the gas phase, as well as in an aqueous medium, and the barriers to transition to different low‐energy conformers are thermally allowed. The molecular electrostatic potential surfaces of the various low‐energy conformers of this molecule have been compared with the related non‐aged enzyme‐bound organophosphorus structure to investigate the role of the calculated conformers on the biological activity of this molecule. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2006

Cadherin Mechanics and Complexation: The Importance of Calcium Binding

E-cadherins belong to a family of membrane-bound, cellular adhesion proteins. Their adhesive properties mainly involve the two N-terminal extracellular domains (EC1 and EC2). The junctions between these domains are characterized by calcium ion binding sites, and calcium ions are essential for the correct functioning of E-cadherins. Calcium is believed to rigidify the extracellular portion of the protein, which, when complexed, adopts a rod-like conformation. Here, we use molecular dynamics simulations to investigate the dynamics of the EC1-2 portion of E-cadherin in the presence and in the absence of calcium ions. These simulations confirm that apo-cadherin shows much higher conformational flexibility on a nanosecond timescale than the calcium-bound form. It is also shown that although the apo-cadherin fragment can spontaneously complex potassium, these monovalent ions are incapable of rigidifying the interdomain junctions. In contrast, removal of the most solvent-exposed calcium ion at the EC1-2 junction does not significantly perturb the dynamical behavior of the fragment. We have also extended this study to the cis-dimer formed from two EC1-2 fragments, potentially involved in cellular adhesion. Here again, it is shown that the presence of calcium is an important factor in both rigidifying and stabilizing the complex.

Synthesis and characterization of the iron(III) complexes of tetra-(β,β'-tetramethylene)tetraphenylporphyrin, (TC6TPP)FeCl and (TC6TPP)FeONO2

The synthesis, NMR and EPR spectroscopic investigation as well as two crystal structures of ( TC 6 TPP ) FeX , where X - = chloride and nitrate are reported. The crystal structure of ( TC 6 TPP ) FeCl reveals an almost equal mixture of saddled and ruffled distortion of the porphyrin as judged by the coefficients of the lowest-frequency vibrational modes (calculated from Normal-Coordinate Structural Decomposition), while ( TC 6 TPP ) FeONO 2 is mainly saddled and more distorted overall. This difference in core structure indicates high conformational flexibility of the TC 6 TPP porphyrin ligand. Overall, both ( TC 6 TPP ) FeX structures have smaller deviation from planarity as compared to five coordinate ( OMTPP ) FeCl and ( OETPP ) FeCl . Therefore, the nature and number of peripheral substituents as well as the axial ligand(s) control geometry and conformation of the porphyrins and fine-tune their spectroscopic properties. EPR data (4.2 K) indicate a predominantly high-spin ( S = 5/2, 97.3%) ground state for ( TC 6 TPP ) FeCl and less pure high-spin state ( S = 5/2, 80%) for ( TC 6 TPP ) FeONO 2. The NMR results support an ideally saddled structure or rapid switching between saddled and ruffled conformations of ( TC 6 TPP ) FeX in solution. The flexibility of the porphyrin core was addressed by using dynamic NMR spectroscopy. The following kinetic parameters for ring inversion were obtained: Δ H ‡ = 24(1) kJ . mol −1, Δ S ‡ = −37(3) J . mol −1. K −1 and Δ H ‡ = 36(1) kJ . mol −1, Δ S ‡ = 20(4) J . mol −1. K −1 for ( TC 6 TPP ) FeCl and ( TC 6 TPP ) FeONO 2, respectively. This results in low free energies of activation, Δ G 298‡ = 35(2) and 30(2) kJ.mol−1, respectively, indicating extremely high flexibility of the porphyrin core in solution (kex298 > 4.2 × 106 and 3.8 × 107 s−1).

Conformational and electrostatic potential study of the benztropine analogs at the dopamine transporter

Thirty seven stable structures of benztropine analogs were optimized at HF/6-31G(d) and HF/6-311G(d,p) level, respectively. The conformational, electrostatic potential and substituted effects were calculated to evaluate their contribution to the dopamine transporter binding affinities. The results reveal that diphenylmethoxyl group is the active group; the high binding affinities of benztropine are partly caused by high conformational flexibility of diphenylmethoxyl group; the molecular activity will be obviously changed when benzyl-Hs are substituted, which is due to the changes in acreage and intensity of the negative electrostatic potential areas of diphenylmethoxyl group. These results will be useful in designing novel benztropine ligands as dopamine uptake inhibitors and molecular probes for the dopamine transporter.

Out-of-plane deformability of aromatic systems in naphthalene, anthracene and phenanthrene

The results of computational studies using the MP2/cc-pvdz approximation reveal high conformational flexibility of aromatic systems in naphthalene, anthracene and phenanthrene. In the gas phase all of these molecules possess two different types of out-of-plane deformations which are associated with the distortion of the benzene ring coplanarity and out-of-plane deformation of the separated rings. A change in the relevant C–C–C–C torsion angles of 15° results in an energy increase of less than 1.25 kcal/mol. Therefore, in every unit amount of time, more than 50% of the molecules have significant non-planar geometry. It is demonstrated that the energy of out-of-plane deformations correlates well with changes in the degree of aromaticity of the conjugated system of whole molecules as well as specific rings.

Validation of molecular docking calculations involving FGF-1 and FGF-2.

A predictive relationship between calculated and observed binding affinities for the complexation of ligands to the fibroblast growth factors FGF-1 and FGF-2 based on molecular docking calculations is described. The majority of the ligands examined in this study have high conformational flexibility, and to account for this, multiple conformers were generated for each and subsequently used in flexible docking calculations. Two scoring functions, Gscore and Emodel, were used to quantify the protein:ligand interaction of which the Emodel score showed the best correlation with experimental binding energies. Both scoring functions, however, predicted similar locations for the ligand sulfate groups in the binding site. The van der Waals radii of nonpolar atoms of both the protein and ligand, which modify the effective sizes of both the protein binding site and the ligand, were also systematically altered by factors of 1.0, 0.9, and 0.8 in order to optimize the conditions for predictive docking. Least squares analyses of the Emodel scores against experimental binding energies yielded best r(2) values of 0.91 and 0.83 for FGF-1 and FGF-2, respectively, with slightly lower q(2) values. Optimized scale factor combinations in conjunction with the least squares lines of best fit based on the Emodel function were used to define a predictive model that was tested against ligands not included in the original set. Acceptable predictions of binding affinity were obtained for use in the initial screening of potential leadlike molecules for both FGF-1 and FGF-2.

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL). The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL. The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds. The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.

Influence of arene–arene interactions on the conformation of acyclic molecules:1H NMR and dipole moment experimental results

AbstractConformational analysis of derivatives of 2‐methyl‐1,3‐propanediol is reported. The diol spacer is substituted with different aromatic residues that are linked by ether or ester functionalities. Most of the experimental evidence was obtained by1H NMR spectroscopy through the analysis of the vicinal coupling constants of the two ABX systems of the spacer. It is found that the main factor influencing the conformation of the molecules is the interaction between the aromatic fragments at the ends of the spacer. Thus, when the electronic character of the two residues is similar (e.g.1–3and5), the molecules have high conformational flexibility. On the other hand, when the electronic character of the two residues is different (e.g.4), the molecules possess a conformational bias, having a preference for folded conformations. These results were corroborated by NOEs and computational modelling. The folded conformation of molecules such as4(and others reported previously) may be due to favourably quadrupolar and van der Waals interactions between the unlike aromatic residues. The conformational energies obtained by the1H NMR analysis of the vicinal coupling constants were used for the calculation of the dipole moments of some molecules (1,3,5and6) and excellent agreement with the experimental values was found. Copyright © 2003 John Wiley & Sons, Ltd.Additional material for this paper is available in Wiley Intersciene

Structural and Functional Adaptations to Extreme Temperatures in Psychrophilic, Mesophilic, and Thermophilic DNA Ligases

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in cold-adapted enzymes, the driving force for denaturation is a large entropy change.

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (α-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.

Molecular dynamics study of the influence of calcium ions on the conformation of gelsolin S2 domain

Gelsolin is an actin-severing protein whose action is promoted by Ca 2+ ions and inhibited by binding to lipid phosphoinositides incorporated in the inner leaflet of the plasma membrane inner lipid bilayer. In this study, we carried out molecular dynamics (MD) simulations to investigate the influence of calcium cations on the conformation of gelsolin S2 domain. First, gelsolin S2 domain taken from the crystal structure of apo-gelsolin (PDB code: 1D0N) was subjected to three 1100 ps MD simulations in a periodic water box with the amber 5.0 force field at T=298 K. In the first simulation (S2_Ca 2+) excess concentration of Ca 2+ was applied, in the second one (S2_phys) the concentration of Ca 2+ ions was physiological and in the third one (S2_w) no Ca 2+ ions were added. The results of MD simulations showed high conformational flexibility of the N-terminal part of the S2 domain. S2_w deviated from the starting structure considerably more that S2_phys and S2_Ca 2+ suggesting that Ca 2+ ions stabilize the conformation of the S2 domain of gelsolin.