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At least 415 records · Page 23Linked to original sources
PubMed2000

NMR spectroscopy, molecular dynamics, and conformation of a synthetic octasaccharide fragment of the O-specific polysaccharide of Shigella dysenteriae type 1.

A synthetic octasaccharide fragment (2) of the O-specific polysaccharide (1) of Shigella dysenteriae type 1 has been studied as its methyl glycoside by one- and two-dimensional homo- and heteronuclear NMR spectroscopy. Complete 1H and 13C NMR assignments have been generated, and the 13C spin-lattice relaxation times have been measured for the octasaccharide 2. A congener (6) of this octasaccharide containing one D-galactose residue with a specific 13C label at C-1 has been synthesized and used to measure interglycosidic 13C-1H coupling by the 2D J-resolved 1H NMR method. From the NMR data, three types of conformational restraints were developed: (a) 29 inter-residue, distance restraints; (b) 48 intra-residue, ring atom dihedral angle restraints, and (c) one heteronuclear, inter-residue dihedral angle restraint. The use of these restraints in a restrained molecular dynamics computation with simulated annealing yielded a conformation resembling a short, irregular spiral, with methyl substituents on the exterior.

Carbohydrate Conformation↗
PubMed2001

MD simulations of homomorphous PNA, DNA, and RNA single strands: characterization and comparison of conformations and dynamics.

MD simulations of homomorphous single-stranded PNA, DNA, and RNA with the same base sequence have been performed in aqueous solvent. For each strand two separate simulations were performed starting from a (i) helical conformation and (ii) random coiled state. Comparisons of the simulations with the single-stranded helices (case i) show that the differences in the covalent nature of the backbones cause significant differences in the structural and dynamical properties of the strands. It is found that the PNA strand maintains its nice base-stacked initial helical structure throughout the 1.5-ns MD simulation at 300 K, while DNA/RNA show relatively larger fluctuations in the structures with a few local unstacking events during -ns MD simulation each. It seems that the weak physical coupling between the bases and the backbone in PNA causes a loss of correlation between the dynamics of the bases and the backbone compared to the DNA/RNA and helps maintain the base-stacked helical conformation. The global flexibility of a single-stranded PNA helix was also found to be lowest, while RNA appears to be the most flexible single-stranded helix. The sugar pucker of several nucleotides in single-stranded DNA and RNA was found to adopt both C2'-endo and C3'-endo conformations for significant times. This effect is more pronounced for single strands in completely coiled states. The simulations with single-stranded coils as the initial structure also indicate that a PNA can adopt a more compact globular structure, while DNA/RNA of the same size adopts a more extended coil structure. This allows even a short PNA in the coiled state to form a significantly stable nonsequentially base-stacked globular structure in solution. Due to the hydrophobic nature of the PNA backbone, it interacts with surrounding water rather weakly compared to DNA/RNA.

Base Sequence↗
PubMed1984

Conformational and dynamic differences between N. meningitidis serogroup B and C polysaccharides, using n.m.r. spectroscopy and molecular mechanics calculations.

1H-N.m.r. spectroscopy has been used to determine the conformation in aqueous solution of the sialic acid residues of the N. meningitidis serogroup B and non-O-acetylated (O-Ac-)-C polysaccharides, and of N-acetylneuraminic acid (NeuNAc). In all cases, the sugar adopts the 2C5 conformation. The side-chain of NeuNAc adopts a conformation such that H-7 and H-8 are approximately anti-periplanar. This conformation is also found in the (O-Ac-)-C polysaccharide, whereas H-7 and H-8 are gauche in the B polysaccharide. Molecular mechanics calculations have been used to probe the conformational preferences of the variously linked sialic acid residues, and the results are in general agreement with those based on the 1H-n.m.r. data. The 13C-n.m.r. spin-lattice relaxation-times have been interpreted in terms of the molecular dynamics of the B and (O-Ac-)-C polysaccharides. Molecular correlation times have been calculated and details of internal rotational or segmental motion elucidated. The C polysaccharide is characterised by internal or segmental motion in the C-7 to C-9 side-chain of the sialic acid repeating-unit, whereas the B polysaccharide has little or no such movement and tumbles in solution as a rigid species with internal rotation of only the pendant C-9 group. The conformational differences suggest a substantially different three-dimensional structure in solution for these polysaccharides.

Magnetic Resonance Spectroscopy↗
PubMed1991

N.m.r. determination of the solution conformation and dynamics of the A.G mismatch in the d(CGCAAATTGGCG)2 dodecamer.

A.G base-paired mismatches that occur during replication are among the most difficult to detect by repair enzymes. Such purine.purine mispairs can exist in two conformations, one of which is stabilized by protons [Gao & Patel (1988) J. Am. Chem. Soc. 110, 5178-5182]. We have undertaken a 1H-n.m.r. and 31P-n.m.r. study of the mismatched dodecamer d(CGCAAATTGGCG)2 as a function of both temperature and pH to determine the conformational features of the A.G mismatch. At pH greater than 7 the mispaired bases are each in the anti conformation and are stacked in the B-like helix. As the pH is decreased, a second conformation becomes populated (apparent pKa approx. 5.9) with concomitant changes in the chemical shifts of protons of the mispaired bases and their nearest neighbours. Data from two-dimensional nuclear-Overhauser-enhancement spectroscopy show unequivocally that, at low pH, the dominant conformation is one in which the mismatched G residues are in the syn conformation and are hydrogen-bonded to the A residues that remain in the anti conformation. Residues not adjacent to the A.G sites are almost unaffected by the transition or the mispairing, suggesting considerable local flexibility of the unconstrained duplexes. Despite the bulging of the mispaired bases, the conformation of the A(anti).G(anti) duplex is very similar to the native dodecamer, whereas the AH+(anti).G(syn) duplex shows a greater variation in the backbone conformation at the mismatched site. According to the chemical shifts, the duplex retains twofold symmetry in solution. The equilibrium between the syn and anti conformations of G9/G21 is strongly dependent on pH, but only weakly dependent on temperature (delta H approx. 16 kJ.mol-1). The first-order rate constant for the transition is approx. 9 s-1 at 283 K and approx. 60 s-1 at 298 K, with an activation enthalpy of approx. 100 kJ.mol-1. The stabilization of the A(anti).G(syn) conformation by protons is consistent with models invoking N1 protonation of adenine. Using the derived glycosidic torsion angles we have used restrained molecular dynamics to build models of the neutral and protonated d(CGCAAATTGGCG)2 oligomers. The results confirm that the A(anti).G(anti) and AH+(anti).G(syn) conformations are favoured at high pH and low pH respectively, in accord with n.m.r. and single-crystal X-ray data.

Base Sequence↗
PubMed2006

Conformational and dynamical properties of disaccharides in water: a molecular dynamics study.

Explicit-solvent molecular dynamics simulations (50 ns, 300 K) of the eight reducing glucose disaccharides (kojibiose, sophorose, nigerose, laminarabiose, maltose, cellobiose, isomaltose, and gentiobiose) have been carried out using the GROMOS 45A4 force field (including a recently reoptimized carbohydrate parameter set), to investigate and compare their conformational preferences, intramolecular hydrogen-bonding patterns, torsional dynamics, and configurational entropies. The calculated average values of the glycosidic torsional angles agree well with available experimental data, providing validation for the force field and simulation methodology employed in this study. These simulations show in particular that: 1) (1-->6)-linked disaccharides are characterized by an increased flexibility, the absence of any persistent intramolecular hydrogen bond and a significantly higher configurational entropy (compared to the other disaccharides); 2) cellobiose presents a highly persistent interresidue hydrogen bond and a significantly lower configurational entropy (compared to the other disaccharides); 3) persistent hydrogen bonds are observed for all disaccharides (except (1-->6)-linked) and typically involve a hydrogen donor in the reducing residue and an acceptor in the nonreducing one; 4) the probability distributions associated with the glycosidic dihedral angles and psi are essentially unimodal for all disaccharides, and full rotation around these angles occurs at most once or twice for (never for psi) on the 50-ns timescale; and 5) the timescales associated with torsional transitions (except around and psi) range from approximately 30 ps (rotation of hydroxyl groups) to the nanosecond range (rotation of the lactol and hydroxymethyl groups, and around the omega-glycosidic dihedral angle in (1-->6)-linked disaccharides).

Carbohydrate Sequence↗
PubMed2001

Conformational stabilization and crystallization of the SecA translocation ATPase from Bacillus subtilis.

SecA is the peripheral membrane-associated subunit of the enzyme complex 'preprotein translocase' which assists the selective transport of presecretory proteins into and across bacterial membranes. The SecA protein acts as the molecular motor that drives the translocation of presecretory proteins through the membrane in a stepwise fashion concomitant with large conformational changes accompanying its own membrane insertion/retraction reaction cycle coupled to ATPase activity. The high flexibility of SecA causes a dynamic conformational heterogeneity which presents a barrier to growth of crystals of high diffraction quality. As shown by fluorescence spectroscopy, the T(m) of the endothermic transition of cytosolic SecA from Bacillus subtilis is shifted to higher temperatures in the presence of 30% glycerol, indicating stabilization of the protein in its compact membrane-retracted conformational state. High glycerol concentrations are also necessary to obtain three-dimensional crystals suitable for X-ray diffraction analysis, suggesting that stabilization of the conformational dynamics of SecA may be required for effective crystallization. The SecA crystals grow as hexagonal bipyramids in the trigonal space group P3(1)12; they possess unit-cell parameters a = 130.8, b = 130.8, c = 150.4 A at 100 K and diffract X-rays to approximately 2.70 A using a high-flux synchrotron-radiation source.

Adenosine Triphosphatases↗
PubMed2006

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature.

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.

Amino Acid Sequence↗
PubMed2003

Hetaryleneaminopolyols and hetarylenecarbopeptoids: a new type of glyco- and peptidomimetics. Syntheses and studies on solution conformation and dynamics.

Ready access to a new class of oligomers has been demonstrated by the synthesis of hetaryleneaminopolyols and hetarylenecarbopeptoids using 3-hydroxymethyl-5-(4-amino-4-deoxy-d-arabinotetritol-1-yl)-2-methylfuran and 5-(4-amino-4-deoxy-d-arabinotetritol-1-yl)-2-methyl-3-furoic acid as novel scaffolds. The conformational behavior of peptidomimetics 22, 23, 25, 26, and 36 have been analyzed by NMR spectroscopy and extensive molecular dynamics simulations. MD simulations using the GB/SA continuum solvent model for water and the MM3 force field provide a population distribution of conformers which satisfactorily agrees with the experimental NMR data for the torsional degrees of freedom of the molecule.

Amino Acids↗
PubMed2001

Conformation and dynamics of normal and damaged DNA.

The genetic information that determines the structure and function of living organisms is encoded in the nucleotide sequence of double-stranded DNA molecules. Despite an apparent structural homogeneity displayed by DNA, subtle local variations in structure and dynamics are functionally significant. Short sequences exhibit specificity for regulatory and catalytic proteins, which mediate fundamental processes necessary to the survival of the cell. However, the molecular basis for specific recognition is still incompletely understood. The "indirect readout" mechanism suggests that the relative propensity of DNA to undergo structural deformations induced by the protein contributes to specific protein-DNA recognition. Although the hypothesis was originally formulated to explain recognition of specific nucleic acid sequences by DNA-binding proteins, it may have particular application to the recognition of DNA damage, because damaged sites in DNA have different equilibrium structure and dynamics from undamaged DNA. In this work, we review the approaches that we took to investigate the questions of sequence- and damage-dependent structure and dynamics of DNA. We describe a statistical thermodynamic model that relates DNA configurational flexibility to sequence-specific protein-DNA binding. The model provides a theoretical basis for interpreting experimental measurements of DNA dynamics. We describe results from MCSCF calculations of the excited states of 2-aminopurine (2AP), which provide the theoretical basis for the intramolecular mechanism of quenching as well as the effect of environment on this process. We then describe our investigations of the effect of stacking, base pairing, and base dynamics on the fluorescence of 2-AP in model systems, which allow us to develop the relationships between steady-state and time-resolved fluorescence parameters on the one hand and local structural and dynamic properties of DNA on the other hand. Finally, we describe the application of the experimental approach to study the conformational heterogeneity of DNA abasic sites, a commonly occurring type of DNA damage. We demonstrate the power of the experimental algorithm to characterize the physical differences between undamaged and damaged DNA, as well as the effects of nucleic acid sequence in both of these contexts. Thus, the work described herein comprises a combination of theoretical and experimental approaches to the problem of sequence- and damage-dependent DNA deformation.

2-Aminopurine↗
PubMed1999

Binding interactions and conformational changes induced by sulfonated aluminum phthalocyanines in human serum albumin.

Phthalocyanines (Pc), which are extensively studied as tumor localizing photosensitizers for photodynamic therapy, are transported by the blood circulatory system to target tissues. Binding interactions between human serum albumin and differently sulfonated aluminum phthalocyanines (AlPcSn; n = 1-4) were studied using optical and ESR spectroscopy. AlPcSn (n = 1-3) occupy one strong binding site and eight weaker sites. The high affinity binding site interactions differ with respect to the degree of sulfonation and isomeric composition of the Pc. Phthalocyanines without SO-3 groups on adjacent iso-indole rings exhibit a high affinity binding site constant of K approximately 3-4 x 10(7) M-1, while Pc with two or three adjacent SO-3 groups show binding for this high affinity site that is no longer independent, but cooperative (alpha = 2), with K approximately 2-6 x 10(6) M-1. Binding isotherms for AlPcS4 and its close analog, tempoyl spin-labeled SL-AlPcS3, do not approach saturation at high ligand concentrations. Competition analyses between AlPcSn and spin-labeled fatty acids (5- and 16-doxyl stearate isomers) reveal that all compounds participate in cooperative (allosteric) interactions with the high affinity binding site of 16-DS, while extruding 5-DS isomer from certain sites and increasing the binding affinity for the remaining. Protein conformational dynamics was studied by ESR spectroscopy using covalent (alkylation of Cys34 residue) and noncovalent spin labeling (employing SL-AlPcS3). Phthalocyanines perturb conformational dynamics parameters (tauc and S) depending on the degree of sulfonation and isomeric composition corresponding to the type of sites, i.e., independent or cooperative, occupied on the HSA molecule.

Allosteric Regulation↗
PubMed1993

Preferred conformations and dynamics of five core structures of mucin type O-glycans determined by NMR spectroscopy and force field calculations.

Glycosyltransferases acting on O-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes and O-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Gal beta (1-3)GalNAc alpha Bzl (1, core 1), GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha Bzl (2, core 2), GlcNAc beta (1-3)GalNAc alpha Bzl (3, core 3), GlcNAc beta (1-6)[GlcNAc beta (1-3)]GalNAc alpha Bzl (4, core 4), GlcNAc beta (1-6)GalNAc alpha Bzl (5, core 6), the elongated core 2, Gal beta (1-4)GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha pNp (6) and GalNAc alpha-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1-6) linkage is much more flexible than the (1-3) or the (1-4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additional N-acetyl group in (3). The conformational equilibrium of beta (1-3) glycosidic bonds of 1 and 3 was not affected by attaching a beta (1-6) linked GlcNAc unit to the GalNAc residue in 2 and 4. However, experimental and theoretical data show that the beta (1-6) linkages of the trisaccharides 2 and 4, which carry an additional beta (1-3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1-6) linkage of 2, 4, and 6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of approximately 4 kcal mol-1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.

Carbohydrate Conformation↗
PubMed1997

Conformational and dynamic differences between N-ras P21 bound to GTPgammaS and to GMPPNP as studied by NMR.

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational changes between the GMPPNP form of human N-ras P21 as compared to GDP and GTPgammaS forms. Full-length N-ras P21 was also compared with protein truncated beyond residue 167, to search for interaction points between the more invariant part of the protein and the variable C-terminal section. In both cases, the reporter was the 15N-H 2D spectrum of aspartate amide groups labeled with 15N. Small truncation-induced changes were seen in the spectrum at the resonances of Asp-54, -108, and -109 which are not far from the C-terminal and, surprisingly, at Asp-57 which is more remote. The spectrum obtained for the GMPPNP-ligated form is similar to that of the GTPgammaS form, except that peaks of several residues are weak at low temperature, and strongly temperature-dependent in their intensity, and a new resonance appears at 15 degrees C and above. The observations are discussed in terms of a two-state model for the GMPPNP-ligated protein, previously proposed by Geyer et al. [(1996) Biochemistry 35, 10308-10320].

Guanosine 5'-O-(3-Thiotriphosphate)↗
PubMed2002

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies.

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.

Aspergillus oryzae↗
PubMed2005

Multiple time scale dynamics of distance fluctuations in a semiflexible polymer: a one-dimensional generalized Langevin equation treatment.

Time-dependent fluctuations in the distance x(t) between two segments along a polymer are one measure of its overall conformational dynamics. The dynamics of x(t), modeled as the coordinate of a particle moving in a one-dimensional potential well in thermal contact with a reservoir, is treated with a generalized Langevin equation whose memory kernel K(t) can be calculated from the time-correlation function of distance fluctuations C(t) identical with x(0)x(t). We compute C(t) for a semiflexible continuum model of the polymer and use it to determine K(t) via the GLE. The calculations demonstrate that C(t) is well approximated by a Mittag-Leffler function and K(t) by a power-law decay on time scales of several decades. Both functions depend on a number of parameters characterizing the polymer, including chain length, degree of stiffness, and the number of intervening residues between the two segments. The calculations are compared with the recent observation of a nonexponential C(t) and a power law K(t) in the conformational dynamics within single molecule proteins [Min et al., Phys. Rev. Lett. 94, 198302 (2005)].

Journal Article↗
PubMed1999

Structural properties of the adipocyte lipid binding protein.

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.

Amino Acid Sequence↗