bluuebunny/crossref_metadata_2025 huggingface dataset:
A molecular dynamics study of thermodynamic and structural aspects of the hydration of cavities in proteins
Authors: Wade Rebecca C. , Mazor Michael H. , McCammon J. Andrew , Quiocho Florante A.
Published: December 2004

The structure and activity of a protein molecule are strongly influenced by the extent of hydration of its cavities. This is, in turn, related to the free energy change on transfer of a water molecule from bulk solvent into a cavity. Such free energy changes have been calculated for two cavities in a sulfate‐binding protein. One of these cavities contains a crystallo graphically observed water molecule while the other does not. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each of the cavities from molecular dynamics simulations of two separate events: the removal of a water molecule from pure water, and the introduction of a water molecule into each protein cavity. From the simulations for the pure water system, the excess chemical potential of water was computed to be −6.4 ± 0.4 kcal/mol, in accord with experiment and with other recent theoretical calculations. For the protein cavity containing an experimentally observed water molecule, the free energy change on hydrating it with one water molecule was calculated as −10.0 ± 1.3 kcal/mol, indicating the high probability that this cavity is occupied by a water molecule. By contrast, for the cavity in which no water molecules were experimentally observed, the free energy change on hydrating it with one water molecule was calculated as 0.2 ± 1.5 kcal/mol, indicating its low occupancy by water. The agreement of these results with experiment suggests that thermodynamic simulation methods may become useful for the prediction and analysis of internal hydration in proteins.
Topographical requirements for δ‐selective opioid peptides
Authors: Nikiforovich Gregory V. , Hruby Victor J. , Prakash Om , Gehrig Catherine A.
Published: December 2004

The conformational possibilities of three different δ‐selective opioid peptides, which are DPDPE magnified image, and DRE (Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, dermenkephalin), were explored using energy calculations. Sets of low‐energy conformers were obtained for each of these peptides. The sets consisted of 61 structures for DPDPE, 32 for DCFPE, and 38 for DRE, including various types of rotamers of the Tyr and Phe side‐chain groups. Comparison of the geometrical shapes of the conformers was performed for these sets using topographical considerations, i.e., examination of the mutual spatial arrangement of the N‐terminal α‐amino group, and of the Tyr and Phe side‐chain groups. The results obtained suggest a model for the δ‐receptor‐bound conformer(s) for opioid peptides. The model suggests the placement of the Phe side chain in a definite position in space corresponding to the g− rotamer of Phe for peptides containing Phe4 and to the t rotamer for peptides containing Phe.3 The position of the Tyr1 side chain cannot be specified so precisely. The proposed model is in a good agreement with the results of biological testing of β‐Me‐Phe4‐substituted DPDPE analogues that were not considered in the process of model construction.
Molecular dynamics simulation in vacuo and in solution of cyclolinopeptide A: A conformational study
Authors: Saviano Michele , Aida Misako , Corongiu Giorgina
Published: December 2004

The conformation of cyclolinopeptide A [c‐(Pro‐Pro‐Phe‐Phe‐Leu‐Ile‐Ile‐Leu‐Val)], a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of molecular dynamics simulations in various molecular environments. Structural and dynamical properties have been analyzed and compared with those experimentally determined. A detailed analysis of hydrogen bonds is reported.
A molecular dynamics simulation of polyalanine: An analysis of equilibrium motions and helix–coil transitions
Authors: Daggett Valerie , Kollman Peter A. , Kuntz Irwin D.
Published: December 2004

An understanding of helix dynamics can aid in interpreting the motions of proteins. The conformational transitions that occur also appear to play a role in protein folding. Structural studies of isolated peptides in solution are just becoming available. However, detailed analysis of the helix–coil transition is still not available and will be difficult to obtain experimentally. For these reasons, we performed a long molecular dynamics simulation of polyalanine at high temperature. Using this approach, we obtain a description of the overall structure and inherent flexibility of the chain as well as a structural picture of the conformational changes that occur. In this way, we can address both equilibrium properties of the peptide and the dynamics and mechanism of the structural transitions. Our results correlate fairly well with the available experimental data and previous simulations aimed at addressing α‐helix dynamics. The peptide spends the bulk of its time fluctuating between different conformations with intermediate helix contents. Transitions between highly ordered and highly disordered structures were rare, but they occurred rapidly. Our distribution of conformations favored collapsed states. Hence, our transitions to structures with high helical content were from fluctuating compact structures. The conversion between helix and coil occurred sequentially on a residue‐by‐residue basis. However, there was local cooperativity; the transition of a residue to the coil state was facilitated after a neighboring group became non‐helical. The relevance of our results to protein folding is also discussed.
On the mechanism of dielectric relaxation in aqueous DNA solutions
Authors: Saif B. , Mohr R. K. , Montrose C. J. , Litovitz T. A.
Published: December 2004

The complex dielectric response of calf thymus DNA in aqueous saline solutions has been measured from 1 MHz to 1 GHz. The results are presented in terms of the relaxation of the incremental contributions to the permittivity and conductivity from the condensed counterions surrounding the DNA molecules. Measurements of the low‐frequency conductivity of the samples also lends support to the condensed counterion interpretation.
β‐Alanyl‐β‐alanine in cyclic β‐turned peptides
Authors: Blasio B. Di , Lombardi A. , Yang X. , Pedone C. , Pavone V.
Published: December 2004

In the present paper we describe the synthesis, purification, and single crystal x‐ray analysis of the cyclic pentapeptide cyclo‐(L‐Pro‐L‐Pro‐L‐Phe‐β‐Ala‐β‐Ala). The peptide was synthesized by classical solution methods and the cyclization of the free pentapeptide was accomplished in good yields in diluted methylenechloride solution using N,N‐dicyclohexylcarbodiimide. The compound crystallizes in the monoclinic space group P21 from hot water with five solvent molecules. The Pro1‐Pro2 peptide bond is cis and the molecular conformation is stabilized by an intramolecular hydrogen bond between the CO group of the β‐Ala5 and the NH of the Phe3 residue. The Pro1‐Pro2 segment occupies the relative positions 2 and 3 of a type VIa β‐turn, while the L‐phenylalanyl‐β‐alanyl‐β‐alanine segment is incorporated in a C13‐like ring structure. The crystal packing is characterized by a network of 11 intermolecular hydrogen bonds involving all the remaining CO, NH, and the water molecules.
Thermally induced conformational transition of double‐stranded xanthan in aqueous salt solutions
Authors: Kitamura Shinichi , Takeo Kenichi , Kuge Takashi , Stokke Bjørn T.
Published: December 2004

The thermally induced conformational transition of double‐stranded xanthans (degree of pyruvate substitution, DSP = 0.45) having Mw = 3.1, 5.7, and 20.3 × 105 has been studied in aqueous salt solutions by high‐sensitivity differential scanning calorimetry (DSC). The double strandedness of these samples in the ordered conformation was ascertained by the value of mass per unit length, ML = 2090 ± 270 g mol−1 nm−1, which was determined from the contour length obtained by electron microscopic observations and the molecular weight by light scattering measurements. The temperature at half completion of the transition T½ for these samples increased linearly with the logarithm of the cation (Na+, K +) concentration. The plot of 1/T½ vs the natural logarithm of cation (Na + ) concentration in mM for the sample with Mw = 5.7 × 105 (15‐SX) yielded the equation 103/T½ = 3.45−0.159 in[Na +]. The specific enthalpy Δhcal, for 15‐SX, essentially independent of salt concentration above 20 mM, was 8.31 ± 0.39 J/g (SD, n = 6). No systematic dependence of molecular weight on the transition temperature and the enthalpy was observed. Application of the Manning polyelectrolyte theory to the system using the DSC data suggested that the separation of the double strand of xanthan into two single chains was not completed at the temperature where the endothermic peak was finished. This suggestion is consistent with recent findings by light scattering measurements as a function of temperature. Our DSC study was extended to include four other samples from various sources. It was found that T½ and Δhcal depend on the pyruvate contents of the samples. For example, the t½(t½/° C = T½/K − 237.15) values for samples with high pyruvate content (DSp = 0.9) and depyruvated (DSp = 0.14) in 20 m Maqueous NaCl were 48.8 and 85.3°C, respectively. Two other samples showed relatively broad DSC curves having shoulders, which were resolved into two independent components. Thermodynamic parameters for each component were examined as a function of salt concentration, and the results obtained were interpreted in terms of the heterogeneity of the pyruvate content of the samples.
Determining molecular weight distributions of antigen–antibody complex by quasi‐elastic light scattering
Authors: Murphy Regina M. , Yarmush Martin L. , Colton Clark K.
Published: December 2004

Physiological properties of soluble antigen–antibody (Ag‐Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag‐Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi‐elastic light scattering data to estimate molecular weight distributions of model Ag‐Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod–Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.
Dependence of the hydration shell structure in the minor groove of the DNA double helix on the groove width as revealed by monte carlo simulation
Authors: Teplukhin A. V. , Poltev V. I. , Chuprina V. P.
Published: December 2004

The hydration shell of several conformations of the polynucleotides poly(dA) · poly(dT), poly (dA) · poly (dU), and poly (dA‐dl) · poly (dT‐dC) has been simulated using the Monte Carlo method (Metropolis sampling). Calculations have shown that the structure of the hydration shell of the minor groove greatly depends on its width. In conformations with a narrowed minor groove, the first layer of the hydration shell of this groove has only one molecule per nucleotide pair that forms H bonds with purine N3 of one pair and pyrimidine O2 of the next pair. The second layer of the hydration shell of such conformations contains molecules that form H bonds between two adjacent molecules of the first layer. The probability of formation of hydration spine is about 20% while the bridges of the first layer are formed with a probability of about 70%.

In the first layer of the minor groove of the B–DNA conformation with wide minor groove there are approximately two water molecules per base pair that form H bonds with purine N3 or pyrimidine O2 and with the sugar ring oxygen of the adjacent nucleotide. The probability of simultaneous H bonding of a water molecule with N3 (or O2) and O of sugar ring is about 30%.

The results of simulation suggest that hydration spine proposed for the narrowed minor groove of oligonucleotide crystals [H. R. Drew, and R. E. Dickerson (1981) Journal of Molecular Biology, Vol. 151, pp. 535–556] can be formed in fibers of poly (dA) · poly (dT), poly (dA) · poly (dU), and poly (dA‐dl) · poly (dT‐dC) as well as in DNA fragments of these sequences in solution. The role of the hydration shell of the minor groove of DNA double helix in stabilization of certain B‐family conformations is discussed.
Anti‐insulin antibody structure and conformation. II. Molecular dynamics with explicit solvent
Authors: Tanner John J. , Nell Laura J. , McCammon J. Andrew
Published: December 2004

Molecular dynamics at 300 K was used as a conformation searching tool to analyze a knowledge‐based structure prediction of an anti‐insulin antibody. Solvation effects were modeled by packing water molecules around the antigen binding loops. Some loops underwent backbone and side‐chain conformational changes during the 95‐ps equilibration, and most of these new, lower potential energy conformations were stable during the subsequent 200‐ps simulation. Alterations to the model include changes in the intraloop, main‐chain hydrogen bonding network of loop H3, and adjustments of Tyr and Lys side chains of H3 induced by hydrogen bonding to water molecules. The structures observed during molecular dynamics support the conclusion of the previous paper that hydrogen bonding will play the dominant role in antibody‐insulin recognition. Determination of the structure of the antibody by x‐ray crystallography is currently being pursued to provide an experimental test of these results. The simulation appears to improve the model, but longer simulations at higher temperatures should be performed.
Quantum yield for preferential photodimerization in long pyrimidine tracts
Authors: Texter John
Published: December 2004

The quantum yield for cyclobutyl‐pyrimidine dimerization in DNA has been observed to increase approximately linearly with increasing pyrimidine tract length. A model without adjustable parameters for this yield is proposed based on energy delocalization, vibronic symmetry switching, and saturation statistics that describe the average number of (base pairwise) breathing modes in a given tract of pyrimidines. This average number of modes is an approximately linear function of the tract length. Monte Carlo techniques are used to simulate base sequences and photochemical events, and indicate that this model is consistent with experiment for Tetrahymena pyriformis DNA.
Molecular dynamics study of the conformational behavior of a representative elastin building block: Boc‐Gly‐Val‐Gly‐Gly‐Leu‐Ome
Authors: Lelj F. , Tamburro A. M. , Villan V. , Grimaldi P. , Guantieri V.
Published: December 2004

The conformational behavior of the synthetic peptide, Boc‐Gly‐Val‐Gly‐Gly‐Leu‐OMe, containing the X‐Gly‐Gly and Gly‐Gly‐X (X = Val or Leu) repeating sequences and constituting a fragment of elastin was investigated by molecular mechanics and molecular dynamics (MD) simulation. The results suggest that, irrespective of the approximations used, the molecule shows a manifold of low energy conformations characterized by γ‐turns and type II β‐turns. Furthermore, MD simulations point out a conformational floppiness due to very low barriers between different conformations. Experimental CD measurements in a virtually apolar medium (dioxane − ε = 2.209), which better mimics the vacuum conditions of the simulation, support the theoretical results. The general emerging picture, indicating the molecule as characterized by a combination of flexibility with conformational preferences, is in agreement with previous experimental findings and enriches of new aspects the description of the microscopic behavior of this molecule suggesting more detailed interpretation of previous data.
Helix formation in poly(Nε,Nε,Nε‐trimethyl‐L‐lysine) and poly(L‐lysine): Dependence on concentration and molecular weight
Authors: Bello Jake
Published: December 2004

Helix formation in (Lys)n · HClO4 and poly(Nε, Nε, Nε–trimethyl‐L‐lysine) · HClO4 [(LysMe3)n · HClO4] is dependent on peptide concentration and on molecular weight. For (LysMe3)n · HClO4 of degree of polymerization (DP) 2510 the midpoint of the coil‐to‐helix transition is 2 mM and for DP of 190 it is 5 mM. For (Lys)n · HC1O4 the peptide concentration for half‐helix is 30–60 times as high, and is only weakly dependent, if at all, on molecular weight. Helix formation is an intermolecular process. The use of methylated (Lys)n as the perchlorate permits study of the intermolecular coil‐helix transition at low concentration, instead of the high concentration (ca. 1–2M) required for (Lys)n · HBr. At constant peptide concentration helix content increases with added NaClO4. The higher the peptide concentration, the less NaClO4 is needed to induce helix.
Conformations of type 1 and type 2 oligosaccharides from ovarian cyst glycoprotein by nuclear overhauser effect spectroscopy and T1 simulations
Authors: Cagas Perseveranda , Bush C. Allen
Published: December 2004

To probe differences in conformation of the type 1 and type 2 linkages in blood group oligosaccharides, two‐dimensional nuclear Overhauser effect spectroscopy (2D‐NOESY) and 1H T1 data were obtained for two blood group A oligosaccharide alditols containing the type 1 and type 2 linkage. The NOE data were interpreted using a complete relaxation matrix approach. Simulations of NOE and T1 values were made using disaccharide and tetrasaccharide model conformations generated by a systematic variation of the glycosidic dihedral angles ϕ and ψ. NOEs from the amide protons of GlcNAc and GalNAc in the type 1 pentasaccharide alditol were obtained, and simulated in a manner similar to those from carbon‐bound protons. In addition to providing data for determining the conformation of the type 1 linkage from amide proton NOEs of GlcNAc and GalNAc to neighboring residues, amide proton NOEs also yield information on the orientation of the acetamido side chains. The amide NOE data indicated subtle differences in the orientation of the amide side chain of GlcNAc among the A type 1 pentasaccharide alditol and two previously studied blood group oligosaccharides, lacto‐N‐difucohexaose 1 and lacto‐N‐fucopentaose 1. From the NOE and 1H T1 data, and from simple rigid geometry energy calculations, it is concluded that the type 1 and type 2 linkages in the oligosaccharides studied have different conformations and that these conformations are relatively rigid in solution.
Refinement of the thrombin‐bound structure of a hirudin peptide by a restrained electrostatically driven Monte Carlo method
Authors: Ripoll Daniel R. , Ni Feng
Published: December 2004

Energy refinement of the structure of a linear peptide, hirudin56–65, bound to thrombin was carried out using a conformational search method in combination with restrained minimization. Five conformations originated from nmr data and distance geometry calculations having a similar global folding pattern but quite different backbone conformations were used as the starting structures. As a result of this approach, a series of low‐energy conformations compatible with a set of upper and lower bounds of interproton distances determined from transferred nuclear Overhauser effects were found. A comparison among the lowest energy conformations of each run showed that the combination of energy refinement plus distance constraints led to a very well‐defined structure for both the backbone and the side chains of the last 7 residues of the polypeptide. Furthermore, the low‐energy conformations generated with this technique contain a segment of 310‐helix involving the last 5 residues at the COOH terminal end.
Tyrosine‐derived polycarbonates: Backbone‐modified “pseudo”‐poly(amino acids) designed for biomedical applications
Authors: Pulapura Satish , Kohn Joachim
Published: December 2004

Starting from L‐tyrosine (Tyr) and its metabolites desaminotyrosine (Dat) and tyramine (Tym), four structurally related model dipeptides were prepared: Dat‐Tym (neither N‐ or C‐terminus present), Z‐Tyr‐Tym (N‐terminus protected by benzyloxycarbonyl), Dat‐Tyr‐Hex (C‐terminus protected by a hexyl ester group), and Z‐Tyr‐Tyr‐Hex (both N‐ and C‐termini present, protected by benzyloxycarbonyl and hexyl ester, respectively). The model dipeptides were used as monomers in the synthesis of polycarbonates. The polymerization reaction in the presence of either phosgene or triphosgene proceeded via the phenolic hydroxyl groups. Polymers with molecular weights of 105,000–400,000 da (by gel permeation chromatography, relative to polystyrene standards) were obtained. The physicomechanical properties (solubility, mechanical strength, glass transition and decomposition temperature, processibility) of the polymers were determined, and an attempt was made to correlate the polymer properties with the nature of the N‐ and C‐terminus protecting groups. The presence of the urethane bond at the N‐terminus protecting group was found to reduce solubility, ductility, and processibility, probably due to interchain hydrogen bonding. The presence of a C‐terminus alkyl ester group increased solubility and processibility. Thus, the most promising candidate polymer for biomedical applications was obtained from Dat‐Tyr‐Hex, the monomer carrying a C‐terminus protecting group only. Since very similar results had recently been obtained for a series of structurally related polyiminocarbonates, the structure property correlations seem to be generally valid.
The structure of Ro 09‐0198 in different environments
Authors: Kessler H. , Mierke D. F. , Saulitis J. , Seip S. , Steuernagel S. , Wein T. , Will M.
Published: December 2004

The constitution and configuration of Ro 09‐0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09‐0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.
NMR and CD studies of triple‐helical peptides
Authors: Brodsky Barbara , Li Ming‐Hua , Long Cynthia Gwynne , Apigo Josefa , Baum Jean
Published: December 2004

Triple‐helix formation of the peptide (Pro‐Hyp‐Gly)10 was monitored by nmr and CD spectroscopy. The two‐dimensional nmr spectra indicated that the Gly Cα H and Pro CδH proton resonances shift upfield in going from the nonhelical to helical form, while hydroxyproline resonances are unchanged. The integrated areas of the helical and nonhelical resonances could be monitored in the one‐dimensional nmr spectrum, and indicate that in the (Pro‐Hyp‐Gly)10 about 90% of the residues are in a defined triple‐helical conformation. The introduction of a glycine to alanine substitution or the deletion of a single hydroxyproline residue in the stable triple‐helical peptide (Pro‐Hyp‐Gly)10 still allows trimers to be formed, but the trimers show a substantial loss of triple helix and decreased thermal stability compared with (Pro‐Hyp‐Gly)10. Two computer models were generated for the Gly → Ala peptide, one with the Ala side chains packed inside the helix and the other with the region containing the alanines forming a β‐bend that loops out from the helix. The nmr data is more consistent with the latter model.
Langevin dynamics of peptides: The frictional dependence of isomerization rates of N‐acetylalanyl‐N′‐methylamide
Authors: Loncharich Richard J. , Brooks Bernard R. , Pastor Richard W.
Published: December 2004

The rate constant for the transition between the equatorial and axial conformations of N‐acetylalanyl‐N′‐methylamide has been determined from Langevin dynamics (LD) simulations with no explicit solvent. The isomerization rate is maximum at collision frequency γ = 2 ps−1, shows diffusive character for γ ≥ 10 ps−1, but does not approach zero even at γ = 0.01 ps−1. This behavior differs from that found for a one‐dimensional bistable potential and indicates that both collisional energy transfer with solvent and vibrational energy transfer between internal modes are important in the dynamics of barrier crossing for this system. It is suggested that conformational searches of peptides be carried out using LD with a collision frequency that maximizes the isomerization rate (i.e., γ ≈ 2 ps−1). This method is expected to be more efficient than either molecular dynamics in vacuo (which corresponds to LD with γ = 0) or molecular dynamics in solvent (where dynamics is largely diffusive).
Theory of delocalized ionic binding to polynucleotides: Structural and excluded‐volume effects
Authors: Ray Jolly , Manning Gerald S.
Published: December 2004

A previously developed theory for the delocalized binding of ions to polyelectrolytes was restricted to point ions and a structurally rigid polyelectrolyte. For the binding of substances like oligolysines and polyamines to DNA, the restriction to point ions would appear not to be realistic. For the binding of ions to flexible chains like single‐stranded polynucleotides, the restriction to a rigid polyelectrolyte may not be realistic. In this article, we assess the effect of relaxation of these two restrictions. Excluded volume among bound ions is modeled by a hard‐rod potential in the context of the theory of a one‐dimensional fluid. The possibility that a flexible chain folds in some manner in the immediate vicinity of a bound ion is modeled by allowing the mean spacing between charged groups on the polymer to become smaller as the number of bound ions increases. We compare our results with recent data on the binding of a series of oligolysines to single‐stranded polynucleotides, which conflict with the predictions of the original theory of delocalized binding of point ions to rigid polyelectrolytes. Inclusion of excluded volume among bound ions does not significantly improve agreement with the data. Substantial improvement in the level of agreement is obtained when the polyion chain is assumed to be flexible. One of our conclusions is that the excluded‐site description of anticooperativity, which was designed for the binding of ligands to discrete sites on a polymer chain, and which does not include the effect of ionic forces, should not be used in cases of delocalized binding of ions.
Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site
Authors: Whitfield Dennis M. , Choay Jean , Sarkar Bibudhendra
Published: December 2004

As model compounds for Ni(II)‐binding heparin‐like compounds isolated from human kidneys (Templeton, D. M. & Sarkar, B. (1985) Biochem. J. 230 35–42.), we investigated two disaccharides—4‐O‐(2‐O‐sulfo‐α‐L‐idopyranosyluronic acid)‐2,5‐anhydro‐D‐mannitol, disodium salt (1a), and 4‐O‐(2‐O‐sulfo‐α‐L‐idopyranosyluronic acid)‐6‐O‐sulfo‐2,5‐an‐hydro‐D‐mannitol, trisodium salt (1b)—that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high‐performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous‐acid‐derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB‐MS, NMR and HPLC‐PAD. Similarly, two synthetic disaccharides—methyl, 2‐O‐sulfo‐4‐O‐(α‐L‐idopyranosyluronic acid)‐2‐deoxy‐2‐sulfamido‐α‐D‐glucosamine, trisodium salt [IdopA2S(α1,4)GlcNSαMe,2a], and 2‐O‐sulfo‐4‐O‐ (α‐L‐idopyranosyluronic acid)‐2‐deoxy‐2‐sulfamide‐6‐O‐sulfo‐α‐D‐glucosamine, tetrasodium salt [IdopA2S(α1,4) GlcNS6SαMe,2b]—were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn(OAc)2 were analyzed to yield 1:1 Zn(II)‐binding constants of 472 ± 59, 698 ± 120, 8,758 ± 2,237 and 20,100 ± 5,598M−1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.
Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation
Authors: Whitfield Dennis M. , Sarkar Bibudhendra
Published: December 2004

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn (II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S(α1,4) AnManOH, 1a, IdopA2S(α1,4)AnManOH6S, 1b, IdopA2S‐(α1,4) GlcNSαMe, 2a, and IdopA2S(α1,4)GlcNS6SαMe, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated 〈(NOE)〉s, 〈T1〉s, and 〈J〉s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L‐idopyranosiduronate ring and the 4E and 4T3 of the anhydro‐D‐mannitol ring are evaluated as are rotations about the C5–C6 hydroxymethylene of the AnManOH (6S) or GlcNS(6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH (6S) or GlcNS (6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially “frozen” such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH (6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS (6S) residues or the sulfate oxygens of the 6‐sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2a and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(α1,4) GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.
The nature of folded states of globular proteins
Authors: Honeycutt J. D. , Thirumalai D.
Published: December 2004

We suggest, using dynamical simulations of a simple heteropolymer modelling the α‐carbon sequence in a protein, that genetically the folded states of globular proteins correspond to statistically well‐defined metastable states. This hypothesis, called the metastability hypothesis, states that there are several free energy minima separated by barriers of various heights such that the folded conformations of a polypeptide chain in each of the minima have similar structural characteristics but have different energies from one another. The calculated structural characteristics, such as bond angle and dihedral angle distribution functions, are assumed to arise from only those configurations belonging to a given minimum. The validity of this hypothesis is illustrated by simulations of a continuum model of a heteropolymer whose low temperature state is a well‐defined β‐barrel structure. The simulations were done using a molecular dynamics algorithm (referred to as the “noisy” molecular dynamics method) containing both friction and noise terms. It is shown that for this model there are several distinct metastable minima in which the structural features are similar.

Several new methods of analyzing fluctuations in structures belonging to two distinct minima are introduced. The most notable one is a dynamic measure of compactness that can in principle provide the time required for maximal compactness to be achieved. The analysis shows that for a given metastable state in which the protein has a well‐defined folded structure the transition to a state of higher compactness occurs very slowly, lending credence to the notion that the system encounters a late barrier in the process of folding to the most compact structure. The examination of the fluctuations in the structures near the unfolding → folding transition temperature indicates that the transition state for the unfolding to folding process occurs closer to the folded state.
The use of flory–huggins theory in interpreting partitioning of solutes between organic liquids and water
Authors: Holtzer Alfred
Published: December 2004

Solute partitioning data for dilute solutions have almost invariably been interpreted by equating experimental values of –RT In Kx (wherein Kx is the mole fraction partition coefficient) to Δμ∞, the standard Gibbs energy change for solute transfer from one solvent to another. Recently, it has been alleged that this relation is insufficiently general. Instead, the statistical mechanical Flory‐Huggins (FH) theory has been recommended for use, because it is designed to account for disparities in molecular size between solute and solvent. Our examination of the thermodynamics of partitioning shows that: (1) The customary interpretation is not only entirely correct (providing only that the solute is dilute), but is model‐independent. (2) The dilute limit of the FH theory is seen to agree entirely with the usual interpretation of –RT In Kx, once certain misnomers are cleared away. (3) The use of FH theory being urged upon us in fact serves only to extract from Δμ∞; (the latter quite correctly determined as –RT In Kx) the contact part of Δμ∞ in order to obtain information on hydrophobic interactions. Some caveats are cited concerning such use of the FH statistical mechanical model.
Hydration of DNA bases: Analysis of crystallographic data
Authors: Schneider Bohdan , Cohen Dawn , Berman Helen M.
Published: December 2004

We present a systematic analysis of water structure around nucleic acid bases. We have examined 28 crystal structures of oligonucleotides, and have studied the patterns of water around the four bases, guanine, cytosine, adenine, and thymine. The geometries of water positions were calculated up to 4.00 Å from base atoms. We have found conformation‐dependent differences in both the geometry and extent of hydration of the bases.
Conformational states of a TT mismatch from molecular dynamics simulation of duplex d(CGCGATTCGCG)
Authors: Venable Richard M. , Widmalm Göran , Brooks Bernard R. , Egan William , Pastor Richard W.
Published: December 2004

The TT mismatch region in duplex d(CGCGATTCGCG) was studied using a 500‐ps molecular dynamics (MD) simulation in water, and a series of 1‐ps MD simulations and energy minimizations in vacuum. The DNA maintained its duplex structure, although the mismatch region showed significantly higher flexibility than the GC regions. The predominant conformation in the 500‐ps MD simulation involved an average −42° propeller twist between T6 and T′6, and a −22° buckle between A5 and T′7, One hydrogen bond was formed between T6 and T′6, and another between T6 and the O2 of T′7 with both Watson‐Crick hydrogen bonds between A5 and T′7 remaining intact. The minimizations resulted in conformations with the equivalent hydrogen‐bonding pattern, as well as ones with “wobble pair” hydrogen bonds between T6 and T′6. However, the wobble pair conformation was found to be unstable in the water simulation.
Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions
Authors: Clack Beatrice A. , Gray Donald M.
Published: December 2004

In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3. We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single stranded DNA, suggesting that bases are stacked in all of the phages. We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages. From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA. The maximum angles were 63° and 64° for the DNAs of class I phages fd and IKe, respectively. The angles were significantly less, 51° and 49°, for the DNAs of the class II phages Pf1 and Pf3, respectively. Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.
Effects of hydration on scale factors for ab initio force constants. IV. trans and cis N‐methylacetamide
Authors: Williams Robert W.
Published: December 2004

Scaled quantum mechanical force fields have been calculated using the 4‐31G basis for trans and cis conformers of both isolated and water‐solvated N‐methylacetamide (NMA). (1) A single set of scale factors for isolated NMA yields relatively correct predictions of the shifts in vibrational frequencies between the trans and cis conformers. This is also true of a single set of scale factors for trans and cis NMA in water. The total standard deviation between measured and calculated frequencies for trans NMA in both isolated and solvated states is 6 cm−1. This implies that it should be possible to use a single set of scale factors to accurately predict the vibrational spectra of a peptide in a variety of conformational states. (2) The computationally predicted effect of hydration on force constants for the supermolecule NMA · nH2O are generally consistent with the experimentally measured effects of hydration on scale factors. These results indicate that supermolecule calculations can be useful in predicting the effects of hydration on spectra. (3) Three types of scale factors are calculated as follows: (a) first from ab initio calculations on an isolated molecule using frequencies measured from isolated molecules; (b) second from calculations on an isolated molecule using frequencies measured from water‐solvated or otherwise hydrogen‐bonded molecules; (c) and third from supermolecule calculations on a molecule hydrogen‐bonded to water, using frequencies measured from water‐solvated molecules. (4) The third type of scale factors are similar to the first type, for confidently measured modes, even though some of the force constants are very different. This suggests that one set of scale factors may be transferable to both isolated and hydrogen‐bonded molecules, and that the simple representation of hydration used here may be a useful approximation. The second type of scale factors yield accurate frequencies, but they may not be generally transferable.
Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base‐pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14)
Authors: Amaratunga Mohan , Snowden‐Ifft Elizabeth , Wemmer David E. , Benight Albert S.
Published: December 2004

The preparation and characterization of DNA dumbbells that contain the 16 base‐pair duplex sequences 5′G‐C‐A‐T‐A‐G‐A‐T‐G‐A‐G‐A‐A‐T‐G‐C3′ (set 1) and 5′G‐C‐A‐T‐C‐A‐T‐C‐G‐A‐T‐G‐A‐T‐G‐C3 ′(set 2) are reported. The dumbbells of set 1 have the duplex stem nucleated on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops. The dumbbells of set 2 have Tn (n = 2, 4, 8, 10) end loops. For the molecules of set 1, effects of end loop size on the electrophoretic mobility, CD and UV absorbance spectra, and cleavage by restriction enzymes, were investigated. Effects of loop size on the CD spectra and restriction enzyme cleavage of the molecules of set 2 were also examined. Optical melting curves of the molecules of set 1 were collected as a function of sodium ion concentration from 30 to 120 mM. These investigations revealed that as loop size decreases, the electrophoretic mobilities, rates of enzyme cleavage, and optical melting temperatures increase. For end loops with at least three T's the observed increases are inversely proportional to loop size. The behavior of the dumbbell with T2 end loops departs from this linear dependence and is anomalous in every experimental context. For molecules with end loops comprised of at least four T's CD spectra were virtually indistinguishable. However, these spectra differed considerably from the CD spectrum of the T2‐looped molecule. The CD spectrum of the dumbbell with T3 end loops displayed features common to the dumbbells with larger loops and T2 end loops. Thermodynamic evidence that the terminal G · C base pairs (bps) nucleating the T2 end loops were intact was obtained from a comparison of the melting temperature of this molecule with that of a DNA dumbbell containing the 14 central bps of the set 1 duplex sequence linked instead by end loops comprised of the four base sequence, C‐T‐T‐C. The tm of this latter molecule was determined to be 9°C less than that of the former dumbbell assumed to contain a 16‐bp stem and T2 end loops. One‐ and two‐dimensional proton nmr measurements performed on the dumbbell of set 2 with T2 end loops and the same sequence with unpaired T's on the ends also verified formation of the T2 end loops, indicated the duplex residues of the dumbbell were in the B form, and showed that the first 3 bps adjoining the loops are distorted. Results are compared with several recently published relevant studies.
Origins and consequences of ligand‐induced multiphasic thermal protein denaturation
Authors: Shrake Andrew , Ross Philip D.
Published: December 2004

The presence of subsaturating levels of a high‐affinity ligand has been demonstrated both by experiment and calculation to have far‐reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand‐binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand‐binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single‐step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non‐two‐state in character; thus, simple deconvolutton should not be carried out. The effects of nonzero values of ΔCP and dΔCP/dT for denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, are explored also.
Synthesis of new hydrophilic microspheres: Optimized carriers for microparticle‐enhanced nephelometric immunoassays
Authors: Marchand J. , Varcin P. , Riochet D. , Montagne P. , Cuilliere M. L , Duheille J. , Pau B.
Published: December 2004

To optimize antigen–antibody reactions, we have synthesized chemically well‐defined hy‐drophilic microspheres. Proteins or haptens were covalently linked to these carriers. When the microsphere conjugates were agglutinated by the corresponding antiserum, the size of the complex artificially increased during the immunological reaction. After optimizing var‐ious parameters such as the hydrophilic character, repulsion charges, and amount of antigen coupled to the microspheres, we developed a rapid and sensitive immunoassay based on laser light scattering by the complexes.
Conformational studies of anionic melittin analogues: Effect of peptide concentration, pH, ionic strength, and temperature—models for protein folding and halophilic proteins
Authors: Ramalingam Kalaiyarasi , Aimoto Saburo , Bello Jake
Published: December 2004

Melittin (MLT), a 26‐residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge −2), or by screening by salt, it goes over to tetrameric α‐helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobia forces. We have changed the net charge to −6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E‐MLT; net charge −4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E‐MLT (net charges −5 and −6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4 concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine. 4HC1 are less effective in helicizing E‐MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8‐120 μM), but E‐MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold‐ and heat‐induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100°C) monomeric MLT (i.e., MLT at low concentration) or E‐MLT results in a monotonic increase in negative ellipticity. In 1.0M NaCl, E‐MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge–charge and charge‐dipole interactions, and hydrophobic effects. E‐MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do corresponding proteins of non‐halophiles.
Discontinuous volume transitions in ionic gels and their possible involvement in the nerve excitation process
Authors: Tasaki I. , Byrne P. M.
Published: December 2004

Discontinuous volume changes in polymer gels carrying negatively ionized groups were studied by varying the molarities of univalent and bivalent cations in the bathing solution. These studies offer a sound basis for elucidating the origin of rapid swelling and heat production in nerve fibers associated with the process of excitation.