bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein
Authors: Zinkel Sandra S. , Crothers Donald M.
Published: December 2004

We describe a method to determine the magnitude of protein‐induced DNA bends relative to a set of standard A tract bends using comparative gel electrophoresis. The DNA bend of interest was that induced by the catabolite activator protein (CAP), the transcriptional activator protein of the lac operon. The set of comparison molecules contained both bends of known magnitude and a bound CAP. The electrophoretic influence of the bound protein was accounted for by placing its binding site at the end of the molecule where its induced bend has little influence. Standard bends at the DNA center were introduced by incorporating 3–9 A6 tracts at ∼ 10.5 base‐pair phasing. The mobility of these control molecules was compared to the mobility of a test molecule of comparable length containing a central CAP‐induced DNA bend. The CAP bend angle was found to be 5.6 ± 0.3 A tract equivalents, or ∼ 100°, independent of the concentration of the gel used within the range tested. The dependence of gel retardation on DNA end‐to‐end distance was found to break down for A tract bend angles above 120°, corresponding roughly to the angle beyond which the long axis of the molecule is no longer parallel to the end‐to‐end vector. We speculate that this may reflect a switch in the mode of migration of molecules through the gel.
Far‐UV CD spectroscopy, IR spectroscopy, and immunological properties of synthetic peptides, including an amphipathic α‐helical peptide from bovine phospholipase A2
Authors: Osthoff Gernot , Nel Alida J.
Published: December 2004

The immunological properties of bovine phospholipase A2 were investigated in order to locate possible sequential epitopes. Polyvalent antiserum raised against the enzyme was tested for its interaction with synthetic peptides derived from regions adopting hydrogen‐bonded secondary structures within the tertiary structure of phospholipase A2. The conformations of each peptide in various solvents were determined by CD and ir spectroscopy in order to relate immunological to structural properties. It was found that sequential epitopes are absent in this enzyme, but that region 90–109 could be a possible T‐cell epitope through the formation of an amphipathic α‐helix.
Assesment of protein reorientational diffusion in solution by 13C off‐resonance rotating frame spin–lattice relaxation: Effect of polydispersity
Authors: Morgan Courtney F. , Schiliech Thomas , Caines G. Herbert
Published: December 2004

The 13C off‐resonance rotating frame spin‐lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in both model in vitro and in vivo systems. The original formalism of James and co‐workers [(1978) J. Am. Chem. Soc. 100, 3590–3594] was constrained by the assumption of random isotropic reorientational motion of a monodisperse protein population. Here we extend the formalism to include polydispersity. Application is made to the alkaline pH induced association of lysozyme, lysozyme–bovine serum albumin mixtures, and to the phase separation of lysozyme salt–water mixtures induced by low temperature.
The influence of Li+, Na+, Mg2+, Ca2+, and Zn2+ ions on the hydrogen bonds of the Watson–Crick base pairs
Authors: Anwander Eugen H. S. , Probst Michael M. , Rode Bernd M.
Published: December 2004

The interaction of mono‐ and divalent metal ions with the nucleic acid base pairs A:T and G:C has been studied using ab initio self‐consistent field Hartree–Fock computations with minimal basis sets. Energy‐optimized structures of the two base pairs with a final base–base distance of L = 10.35 Å have been determined and were further used in calculations on ternary complexes Mn+ – A:B together with previously computed coordination geometries of the cations at adenine (Ade), thymine (Thy), and guanine (Gua). Besides the binding energy of the various metal ions to the base pairs, changes in the stability of the H bonds between Ade and Thy or Gua and Cyt have been determined. Polarization effects of the metal ion on the ligand turned out to increase the binding between complementary bases. Regardless of the metal species, cation binding to Gua N(3) and Thy O(2) leads to a special increase in H‐bond stability, whereas binding to Ade N(3) changes the H‐bond stability least. Situated in between are the stabilizing effects caused by Gua and Ade N(7) coordination. A remarkable relation between the stability of the H bond and the distance from metal binding site to H bonds was found. This relationship has been rationalized in terms of partial charges of the atoms participating in H bonding, which can reveal the trend in the electrostatic part of total H bond energy. It can be shown that a short distance between coordination site and acceptor hydrogen increases the H‐bond strength substantially, while a long distance shows minor effects as supposed. On the other hand, the opposite effect is observed for the influence of the distance between binding site and donor atom. A comparison of our findings with a new model of transition metal ion facilitated rewinding of denatured DNA proposed by S. Miller, D. VanDerveer, and L. Marzilli is given [(1985) J. Am. Chem. Soc. 107, 1048–1055].
Environmental control of reactions: Influence of poly(glutamate) on the reactivity of cysteine‐quaterpyridineiron (III) mixtures
Authors: Paradossi G. , Palleschi A. , Desideri A. , Pispisa B.
Published: December 2004

Quaterpyridineiron(III) complex ions (FeT) anchored to ordered poly(L‐glutamate) or poly(D‐glutamate) were used as enantiomeric systems for stereoselective oxidation of L‐cysteine, a widely encountered ligand in both heme and nonheme iron–sulfur proteins. Surprisingly, electron transfer from substrate to the central metal ion does not take place, despite the favorable thermodyamic driving force. Instead, a stable, polymer‐supported, Fe(III)T‐cysteinate complex forms, to which no stereoselectivity is associated. This finding contrasts the known lability of iron(III)–thiolate complexes, ultimately yielding disulfides and iron(II) species, and is relevant in view of the current interest in model compunds of cytochrome P‐450, where cysteine is axially bound to ferric prophyrin. The formation of this complex was studied by kinetics and low‐temperature electron paramagnetic resonance spectroscopy, while the stereochemical features of the diastereomeric encounter intermediates were investigated by theoretical conformational analysis. The results collectively emphasize the environmental contraol of the polypeptide matrix on the stability of cysteinate sulfur axial ligation. Implications of the absence of stereoselectivity in the reaction investigated are also discussed in the light of a few general considerations concerning chiral discrimination in reactions between optically active compounds.
A molecular dynamics simulation of the (dG)6·(dC)6 minihelix including counterions and water
Authors: Zielinski Theresa Julia , Shibata Masayuki
Published: December 2004

The results of a 60 ps molecular dynamics (MD) simulation of (dG)6·(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to flucturate with time, showing the dynamic nature of the base‐pairing interaction. These fluctuations gave rise to unusual hydrogen‐bonding patterns. Good intrastrad base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B‐DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B‐DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n·(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.
An ellipticine derivative (oxazolopyridocarbazolium) 3′ linked to tetrathymidylate stacks intramolecularly with the nearest thymine at low concentration and head‐to‐tail intermolecularly at high concentration
Authors: Bazile D. , Guittet E. , Piriou J.‐M. , Lebret M. , Paoletti J.
Published: December 2004

The solution conformation of a tetrathymidylate linked through an ester bond to an ellipticine derivative oxazolopyridocarbazolium (OPC) at the 3′ position was investigated using one‐ and two‐dimensional nmr experiments. Since the total electric charge of the OPC ring may influence self‐association, we first determined the pKa of the oxazole cyclic acidic function. Nuclear Overhauser effect spectroscopy experiments showed that, at low concentration, the OPC stacks intramolecularly with the nearest thymine at the 3′ end. At highest concentration, however, the OPC rings are self‐associated. The stacking constant was calculated using 1H chemical shift dilution experiment. The conformational model suggested by P‐nmr was tested by molecular mechanics computations.
The thermodynamics of hydrogen isotope exchange in lysozyme: The influence of glycerol
Authors: Gregory Roger B. , Rosenberg Andreas , Knox Donald , Percy Amy J.
Published: December 2004

The temperature dependence of hydrogen isotope exchange rates for lysozyme in 5 molal aqueous glycerol and for poly (D,L‐alanine) in a range of glycerol concentrations from 0 molal to 8 molal have been determined. The activation enthalpy of base‐catalyzed exchange for poly (D,L‐alanine) in water is 4 kcal/mol and passes through a minimum at about 2 molal glycerol before returning to a value of 4 kcal/mol at 4 molal glycerol. Exchange rates for lysozyme have been analyzed with transition state and Kramers's theories. The activation parameters for exchange of protons in lysozyme in the presence of 5 molal glycerol show a similar qualitative behavior to those determined for exchange in the absence of glycerol [R. B. Gregory et al. (1982) Biochemistry 24, 6523–6530]. The activation enthalpies and entropies for the fast‐exchanging protons show a gentle increase as H(t), the number of hydrogens remaining unexchanged, decreases. By contrast, the activation parameters for the slowest exchanging protons [H(t) < 20] increase dramatically as H(t) decreases. As in water, the activation parameters for exchange of the fast‐ and slow‐exchanging protons in glycerol solution are characterized by two distinct compensation temperatures (510 ± 100 K for the fast protons and 340 ± 40K for the slow protons). These values are not significantly different from those determined for exchange in water.

The activation parameters, ΔH‡, and TΔS‡, are both several kcal/ mol lower for exchange in glycerol solutions compared with water. Enthalpy and entropy changes for the thermal unfolding of lysozyme in glycerol solutions [results of K. Gekko (1982) J. Biochem. 19, 1197–1204] are larger than those determined for unfolding in water. This suggests that exchange does not involve local unfolding of segments of the protein. There is evidence for a contribution from thermal unfolding for exchange of the slowest protons in glycerol solutions which is not observed in water.

Analysis of exchange rates for the fast protons as a function of temperature and viscosity with Kramers' equation provides values of the viscosity exponent. This is found to increase with decreasing values of H(t). These results are not easily rationalized with Gavish's model [(1980) Phys. Rev. Lett. 44, 1160] of a “position‐dependent, internal protein viscosity” and suggest that the Gavish model of the friction coefficient is incorrect. A dissection of the effect of glycerol on exchange of the fast protons into viscosity and thermodynamic contributions is not possible with the present data. Both factors are suggested to influence exchange of the fast protons.
The irreversible binding of 2‐site ligands to hetereogeneous polymers
Authors: Schumaker Mark F. , Epstein Irving R.
Published: December 2004

We obtain an analytical solution for the binding of 2‐site ligands to a heterogeneous lattice. The model also describes the kinetics of chemical reactions between adjacent pendant groups of a copolymer. Our analysis is based on the principle of independence, as formulated by Gonzalez and Hemmer. Several special cases are solved in closed form; a power series solution is developed for the general problem. The results may be modified to take into account fixed total ligand. We also analyze competition between 1‐site and 2‐site ligands. As an illustration of the results, we examine in detail the kinetics and final extent of binding for two special cases.
Peptide design: Influence of a guest Aib‐Pro segment on the stereochemistry of an Oligo‐Val sequence—solution conformations and crystal structure of Boc‐(Val)2‐Aib‐Pro‐(Val)3‐OMe
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Uma K. , Balaram Hemalatha , Balaram P.
Published: December 2004

The peptide Boc‐Val‐Val‐Aib‐Pro‐Val‐Val‐Val‐OMe has been synthesized to investigate the effect of introduction of a strong β‐turn promoting guest segment into an oligopeptide with a tendency to form extended structures. 1H‐nmr studies in solution using analysis of NH group solvent accessibility and nuclear Overhauser effects suggest an appreciable solvent dependence of conformations. In chloroform a 310‐helical structure is favored while in dimethylsulfoxide an Aib‐Pro β‐turn with extended arms on either side is suggested. In the crystal, the backbone forms a somewhat distorted 310‐helix despite the presence of a Pro residue in the middle. Among the four possible intrahelical hydrogen bonds three are of the 4 → 1 type and one 5 → 1. Head‐to‐tail NH ⃛OC hydrogen bonds link the helical molecules into continuous columns. The space group is P212121 with a = 11.320(2), b = 19.889(3), and c = 21.247(3) Å.
Linear dichroism characteristics of ethidium–and proflavine–supercoiled DNA complexes
Authors: Swenberg Charles E. , Carberry Susan E. , Geacintov Nicholas E.
Published: December 2004

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04–0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electroporphoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA‐bound EB molecules, is constant within the range of r values of 0.02–0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple interacalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules. The differences in the LD characteristics of the bound PF and EB molecules may be due to subtle differences in the mechanisms of binding, perhaps reflecting differences in the torsional dynamics and local rigidities in superhelical DNA [Wu et al. (1988) Biochemistry 27, 8128–8144] induced by these two different in tercalating agents.
Protein folding and chain collapse
Authors: Allegra Giuseppe , Ganazzoli Fabio , Bignotti Fabio , Bolognesi Martino
Published: December 2004

It is currently assumed that the folded structure of a globular protein is controlled in a highly deterministic way by its amino acid sequence. We show here that a very different although not necessarily contrasting viewpoint can be adopted. From statistical treatment of x‐ray results, we suggest that the folding pattern essentially follows the collapse to be expected on statistical‐mechanical grounds for an ideal chain effectively experiencing self‐attraction and comprising identical units, whose conformational properties are obtained as an average over the actual amino acid units. The local details of folding of each protein, obviously dictated byits amino acid sequence, can be regarded as statistical fluctuations.

We consider 31 globular fragments belonging to 21 different water‐soluble, nonmembrane proteins. By the theory of chain collapse proposed by two of us [G. Allegra and F. Ganazzoli (1985) J. Chem. Phys. 83, 397], all the average intramolecular distances may be obtained. Accordingly, first an average plot of the mean‐square distances between kth neighboring amino acid units is constructed, starting from the observed crystallographic coordinates. Then the plot is basically reproduced with a wormlike chain model undergoing collapse as a result of intramolecular attractive forces. Agreement is especially good for short amino acid sequences (k ≲ 30), in which case the statistical sampling is more accurate, enabling us to determine the model parameters. The resulting mean‐square radius of gyration is also in good agreement with the experimental average, whereas the unperturbed characteristic ratio is roughly consistent with results from conformational calculations by W. L. Mattice [(1977) Macromolecules 10, 516], based on the rotational isomeric state approach.
Helix aggregation in peptide crystals: Occurrence of either all parallel or antiparallel packing motifs for α‐helices in polymorphs of Boc‐Aib‐Ala‐Leu‐Ala‐Leu‐Aib‐Leu‐Ala‐Leu‐Aib‐OMe
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Uma K. , Balaram P.
Published: December 2004

Three crystalline polymorphs of the helical decapeptide, Boc‐Aib‐Ala‐Leu‐Ala‐Leu‐Aib‐Leu‐Ala‐Leu‐Aib‐OMe, have been obtained. Antiparallel helix aggregation is observed in crystals grown from methanol (A), while completely parallel packing is observed in crystals from isopropanol (B) or an ethylene glycol‐ethanol mixture (C). Crystals B and C are very similar in molecular conformation and packing. The packing motifs in crystals A and B consist of rows of parallel molecules, with an almost identical arrangement in both crystals. In crystal A, adjacent rows assemble with helix axes pointed in oppsite directions, whereas in crystal B all rows assemble with helix axes pointed in the same direction. Electrostatic interactions between helix dipoles do not appear to be a major determinant of packing modes. The structures also do not provide a ready rationalization of packing preferences in terms of side‐chain interactions or solvation. The α‐helix of the peptide in crystal A has seven 5 → 1 hydrogen bonds; the helix in crystal B is a mixed 310/α‐helix. The crystal parameters are as follows. Crystal A: C51H92N10O13·CH3OH, space group P21 with a = 10.498(1) Å, b = 18.189(3) Å, c = 16.475(3) Å, β = 99.28(1)°, Z = 2, R = 9.6% for 1860 data. Crystal B: C51H92N10O13·C3H7OH, space group P21 with a = 10.534(1) Å, b = 28.571(4) Å, c = 11.055(2) Å, β = 95.74(1)°, Z = 2, R = 6.5% for 3251 data. Crystal C: C51H92N10O13·C2H5OH, space group P21, with a = 10.450(1) Å, b = 28.442(5) Å, c = 11.020(2) Å, β = 95.44(1)°, Z = 2, R = 14.8% (isotropic) for 1948 data.
Conformational sampling using high‐temperature molecular dynamics
Authors: Bruccoleri Robert E. , Karplus Martin
Published: December 2004

High‐temperature molecular dynamics as a method for conformational search was explored on the antigen combining site of McPC 603, a phosphorylcholine binding immunoglobulin. Simulations at temperatures of 500, 800, and 1500 K were run for 111.5, 101.7, and 76.3 ps, respectively. The effectiveness of the search was assessed using a variety of methods. For the shorter hypervariable loops, molecular dynamics explored an appreciable fraction of the conformational space as evidenced by a comparison to a simple theoretical model of the size of the conformational space. However, for the longer loops and the antigen combining site as a whole, the simulation times were too short for a complete search. The simulations at 500 and 800 K both generated conformations that minimized to energies 200 kcal/mole lower than the crystal structure. However, the 1500 K simulation produced higher energy structures, even after minimization; in addition, this highest temperature run had many cis‐trans peptide isomerizations. This suggests that 1500 K is too high a temperature for unconstrained conformational sampling. Comparison of the results of high temperature molecular dynamics with a direct conformational search method, [R. E. Bruccoleri & M. Karplus (1987) Biopolymers 26, 137–168]. showed that the two methods did not overlap much in conformational space. Simple geometric measures of the conformational space indicated that the direct method covered more space than molecular dynamics at the lower temperature, but not at 1500 K. The results suggest that high‐temperature molecular dynamics can aid in conformational searches.
The europium (III)‐induced conformational transitions of poly(dG‐dC) · poly(dG‐dC) and poly(dG‐m5dC) · poly(dG‐m5dC) as studied by europium(III) luminescence, UV, and CD spectroscopy
Authors: Klakamp Scott L. , Horrocks William Dew.
Published: December 2004

The Eu3+ ion induces a B to Z transition in poly(dG‐dC) · poly(dG‐dC) and poly(dG‐m5dC) · poly(dG‐m5dC). Other conformations were also elicited by this ion. The conformational changes of these polynucleotides were followed by CD and uv spectroscopy. Eu3+ excitation spectra and Eu3+ excited‐state lifetimes were measured for the Eu3+ ion bound to the duplexes in various conformations. B‐form poly(dG‐dC) and poly(dG‐m5dC) appear to have a single class of the Eu3+ binding site while the Z‐form observed conformation exhibits two. The numbers of H2O molecules coordinated to the Eu3+ ion bound to the various conformations of the duplexes were determined by measuring Eu3+ excited‐state lifetimes in H2O and D2O. The first‐order rate constants for the Eu3− and [Co(NH3)6]3+ induced B to Z transitions were measured. The Eu3+ mediated conversion is much faster than the [Co(NH3)6]3+ induced conversion in both polynucleotides.
Determination of eigenvectors in DNA polymer spectroscopy by isotopic substitution
Authors: Van Zandt L. L. , Saxena V. K.
Published: December 2004

A simple relationship between frequency shifts to be expected in molecular vibrational spectra upon isotopic substitution and component values of eigenvectors is displayed. Application of this expression to the problem of force constant refinement is discussed in terms of a refinement algorithm developed some years ago to overcome particular problems associated with large molecular systems. Calculations of expected isotopic shifts have been calculated and are tabulated here for experimental reference.
Dynamic bending rigidity of DNA
Authors: Song Lu , Schurr J. Michael
Published: December 2004

Rapidly relaxing components in the decay of the transient electric dichroism of DNA restriction fragments were reported by Diekmann et al. [(1982) Biophys. Chem. 15, 263–270] and Pörschke et al. [(1987) Biopolymers 26, 1971–1974]. These are analyzed using a new normal mode theory for weakly bending rods and assigned to bending. The longest bending relaxation times for fragments with 95–250 base pairs coincide with the theoretical curve calculated for a dynamic bending rigidity corresponding to a dynamic persistence length Pd = 2100 Å. Analysis of the relative amplitudes of fast and slow components following weak orienting pulses is also consistent with a rather large dynamic persistence length. The enhancement of the relative amplitude of the fast component in large electric fields is attributed to steady‐state bending of initially perpendicular DNAs by the field. Several reasons are proposed why the dynamic bending rigidity is 4 times larger than the apparent static bending rigidity inferred from equilibrium persistence length measurements on the same fragments.
Anomalously slow electrophoretic mobilities of DNA restriction fragments in polyacrylamide gels are not eliminated by increasing the gel pore size
Authors: Stellwagen Anne , Stellwagen Nancy C.
Published: December 2004

The effect of gel pore size on the anomalous mobility of certain curved DNA restriction fragments in polyacrylamide gels was studied by comparing the electrophoretic mobilities of normal and anomalous fragments in gels of varying composition but constant acrylamide concentration. Molecular weight ladders were prepared from two 147 base pair G‐C rich restriction fragments, called 12A (anomalous) and 12B (normal), obtained from the MspI digestion of plasmid pBR322. The electrophoretic mobilities of multimers of the two fragments increasingly diverged with increasing molecular weight. The anomalous mobility of fragment 12A was essentially independent of gel pore size, regardless of whether the pore size was varied by increasing the acrylamide concentration at constant cross‐linker concentration or by increasing the cross‐linker concentration at constant acrylamide concentration. The anomalous mobility of higher multimers of fragment 12A decreased with increasing gel pore size when the pore size was varied by changing the gel concentration. However, when the gel pore size was changed by varying the cross‐linker concentration at constant acrylamide concentration, the anomalous mobility of higher multimers of fragment 12A went through a maximum and then decreased as the pore size was increased. Copolymerizing acrylamide with high molecular weight linear polyacrylamides had no effect on the anomalous mobility of the 12A multimer ladder, even though the apparent absolute mobilities of all fragments increased markedly. Only by incorporating charged residues into the gel matrix or by copolymerizing acrylamide with the intercalator ethidium bromide could the difference in mobility between the 12A and 12B multimer ladders be substantially reduced or eliminated. Similar results were observed with a molecular weight ladder containing 78 base pair repeats of the bending locus of kinetoplast DNA. These results suggest that pore size alone is not responsible for the anomalously slow migration of curved DNA molecules in polyacrylamide gels.
Bond‐Optimized ring closure for proline: Comparison of conformations and semiempirical energies with small molecule X‐ray structures
Authors: Thomasson Kathryn A. , Applequist Jon
Published: December 2004

A method is described for generating proline ring structures by successive addition of atoms, wherein ring closure is achieved by optimizing the fit to known ring bond‐angles and one closing bond‐length (“bond‐optimized ring closure”). Two ring torsion angles are fixed independently within broad, allowed ranges, and the remaining torsion angles are determined uniquely in most cases. The independent torsion angles are chosen as ϕ and χ2, and ring closure is achieved without prohibitive strain through most of the ranges −130° < ϕ < −20° and −60° < χ2 < 60°. Comparisons of predicted ring structures to 191 X‐ray diffraction structures from the literature, starting with the known values of ϕ and χ2, yielded root‐mean‐square deviations of 4.8° in χ1, 4.7° in χ3, 8.3° in χ4, and 0.3–2% in the ring bond angles and the N‐Cδ distance. Semiempirical energies were calculated for the optimized structures using three sets of energy parameters from the literature. The energy surfaces show broad minima coinciding with the torsion angle regions in which the highest concentrations of observed structures are found. Two of the sets of energy parameters produce double minima corresponding to the “up” and “down” puckered conformations.
Relation between calculated amide frequencies and solution structure in Ala‐X peptides
Authors: Williams Robert W. , Weaver James L. , Lowrey Alfred H.
Published: December 2004

Computational techniques have been used to aid interpretation of observed systematic shifts in the amide III frequencies of Ala‐X peptides. Optimized structures and frequencies have been calculated for Ala‐X peptides using GAUSSIAN86/88 with the 4‐31G basis, MOPAC, and normal mode methods based on empirical force fields. We observe the following: (1) Frequencies calculated using scaled GAUSSIAN86 force constants correlate well with the experimental results. (2) Structures of the Ala‐X peptides optimized by GAUSSIAN show a clear trend toward lower values of the dihedral angle ϕ as the X side chain becomes larger, while structures optimized here using semiempirical and empirical force fields do not show trends. (3) Computational changes in peptide conformations from β‐sheet to α‐helix produce large changes in both amide I and amide III frequencies that are inconsistent with the experimental results. (4) Computational changes in the dihedral angle ϕ of Ala‐Ala produce a change in the amide III frequency consistent with the experimental results. (5) The experimental frequency shifts cannot be attributed directly to the effects of changing mass.
Heteronuclear two‐dimensional 15N‐ and 13C‐nmr studies of DNA oligomers and their netropsin complexes using indirect proton detection
Authors: Ashcroft Joseph , Live David H. , Patel Dinshaw J. , Cowburn David
Published: December 2004

Heteronuclear multispin coherence proton‐detected two‐dimensional nmr spectroscopic experiments were used to obtain information on protonated carbons and nitrogens of the self‐complementary d (G‐G‐T‐A‐T‐A‐C‐C) and d (G‐G‐A‐A‐T‐T‐C‐C) duplexes, with and without the drug netropsin dissolved in aqueous solution. Many correlations of protons coupled to 13C nuclei on the base and sugar rings of the octanucleotides were detected, allowing the carbon resonances to be assigned based on previous homonuclear proton two‐dimensional nmr studies. Imino nitrogen assignments can also be made using the proton assignments from previous one‐dimensional nuclear overhauser effect experiments. Imino nitrogen shifts may be useful indicators of changes in local hydrogen‐bonding interactions to base‐pair edges.
Virtual rigid body dynamics
Authors: Head‐Cordon Teresa , Brooks Charles L.
Published: December 2004

An important direction in biological simulations is the development of methods that permit the study of larger systems and/or longer simulation time scales than is currently feasible by molecular dynamics. One such method designed with this objective in mind is stochastic boundary molecular dynamics (SBMD). SBMD was developed for the characterization of spatially localized processes in proteins, and has been shown to successfully reproduce structural and dynamical properties of these macromolecules, as compared to a molecular dynamics control simulation, when concerted or global motions are not present. The virtual rigid body dynamics method presented in this work extends the range of applicability of the SBMD method, by providing a framework to include these important long time scale conformational transitions. In this paper we describe the two‐step implementation of the virtual rigid body model: first, the reduction of the full atomic representation to a reduced particle (virtual bond) model, and second, the propagation of the dynamics of flexibly connected rigid bodies containing virtual atom sites.
High directional Monte Carlo procedure coupled with the temperature heating and annealing as a method to obtain the global energy minimum structure of polypeptides and proteins
Authors: Shin Jae Kwang , Jhon Mu Shik
Published: December 2004

A high directional Monte Carlo procedure that predicts the topology of the energy hyper‐surface before it walks, is developed as a method to obtain a global energy minimum structure of polypeptides and proteins. It calculates its covariance matrix, which controls the individual trial step distribution of the next set of steps, from the second moment of the actual walk segment in the previous set. The method is successfully applied to the pentapeptide Metenkephalin system. And it is shown that some initial heating process, which provides the more flexible molecule, is necessary in order to overcome the energy barriers that can be overestimated by some biases in the empirical description of the system. The sampling efficiency, traced by an average conformational changes, is found to be at least 20 times greater than the one in the conventional Metropolis Monte Carlo methods, and it is expected that this increases in efficiency will be more prominent when the system is larger.
Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch
Authors: Guo Qiu , Lu Min , Kallenbach N. R.
Published: December 2004

Four‐arm DNA branched junctions are stable analogues of Holliday recombinational intermediates. A number of four‐arm DNA junctions synthesized from oligonucleotides have now been studied. Gel mobility or chemical footprinting experiments on several immobile four‐arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other. We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference. The pattern of self‐protection of the strands of J1c exposed to Fe(II) · EDTA‐induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over. Moreover, the Fe(II) · EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1. The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4°C and in the presence of 10 mM Mg2+, based on chemical reactivity data. This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7. This difference could reflect either sequence‐dependent differences in the equilibrium among isomers, or in the structure of these junctions. Chemical footprinting experiments using the probes MPE · Fe(II) and (OP)2Cu(I) show that the high‐affinity ligand binding site in immobile junctions is determined by junction geometry.
An estimate of the nearest neighbor base‐pair content of 5S RNA using CD and absorption spectroscopy
Authors: Johnson Kenneth H. , Gray Donald M.
Published: December 2004

We analyzed the CD and uv absorption spectra of 5S RNA from Escherichia coli using the method developed in the preceding paper. The analysis of spectra of 5S RNA at 20°C in 0.1 M NaClO4, 2.5 mM Na+ (phosphate), pH 7.0, and 0.5 mM MgSO4 gave 7 ± 3.6 A · U base pairs, 25 ± 3.6 G · C base pairs, and 7.5 ± 3.6 G · U base pairs. Estimates of nearest neighbor base pairs were more consistent with the Pieler–Erdmann and the Gewirth–Moore secondary structure models than with the Fox–Woese or the Burns–Luoma–Marshall models.

We also examined the structure of 5S RNA as a function of temperature. The melting profile exhibited two transitions—one at about 35°C and one above 50°C. Our spectral data showed that helices I and II were stable during the first transition, and agreed with other data that helix III was the most likely helix to have melted. The results from this in‐depth study of 5S RNA indicate that our method of analysis should be useful for studying the secondary structures of other small, unmodified RNAs.
Conformations and time‐resolved fluorescence of oligomers of (−)‐epicatechin with 4β → 8 interflavan bonds
Authors: Cho Donghwan , Porter Lawrence J. , Mattice Wayne L.
Published: December 2004

Oligomers of (−)‐epicatechin with 4β → 8 interflavan bonds, and as many as five monomer units, have been studied by conformational analysis and time‐resolved fluorescence. The conformational analysis yields 2x−1 conformations for each oligomer with x monomer units. There are two conformations accessible at each interflavan bond. These conformations are denoted by + and −. The dominant conformations for the trimer and higher oligomers have an interaction between the phenolic hydroxyl groups on monomers i and i+2. This interaction involves the hydroxyl group at C(5) on monomer i, and either C(13) or C(8) of monomer i+2, depending on whether the conformation of the two intervening interflavan bonds is + + or + −, respectively. Minor contributions to the ensemble for the tetramer and pentamer are made by conformations that contain the sequence of successive interflavan bonds denoted by −+ or −−. In −+ the interaction between monomer units i and i + 2 involves an aliphatic hydroxyl with a phenolic hydroxyl, and there are no hydroxyl‐hydroxyl interactions between units iand i + 2 in −−. The onset of a different decay law for the fluorescence when x increases from 3 to 4 may be associated with the appearance of the −+ and −− conformations as minor constituents in the ensemble.
New insights on μ/δ selectivity of opioid peptides: Conformational analysis of deltorphin analogues
Authors: Tancredi T. , Temussi P. A. , Picone D. , Amodeo P. , Tomatis R. , Salvadori S. , Marastoni M. , Santagada V. , Balboni G.
Published: December 2004

The message domain of dermorphin (Tyr‐D‐Ala‐Phe), a natural μ‐opioid heptapeptide, has long been considered the main cause of the high μ selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolorof deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of μ‐selective peptides, but endowed of an extremely high δ selectivity, the highest of all natural opioid peptides.

A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for μ and δ opioids that recognize the same β‐turn in the N‐terminal part, but discriminate for the conformation and polarity of the C‐terminal part.

Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.
Two‐Dimensional 1H‐nmr study of antigen–antibody interactions: Binding of synthetic decapeptides to an anti‐acetylcholine receptor monoclonal antibody
Authors: Cung Manh Thong , Demange Pascal , Marraud Michel , Tsikaris Vassilios , Sakarellos Constantinos , Papadouli Irene , Kokla Anna , Tzartos Socrates J.
Published: December 2004

Two‐dimensional NMR experiments [correlated spectroscopy (COSY) and two‐dimensional transferred nuclear Overhauser enhancement spectroscopy (TR‐NOESY)] have been applied to study the interactions of a monoclonal antibody (mAb) directed to the main immunogenic region (MIR) of the acetylcholine receptor (AChR), and four synthetic decapeptides from the MIR. The decapeptides were the Torpedo AChR α67‐76 fragment (W 67‐N68‐P69‐A70‐D71‐Y72‐G73‐G74‐I75‐K76) and its three [A69], [A73], and [A76] analogues. The results led to the following conclusions: (1) the magnitude of the TR‐NOE cross peaks does not depend only on the structuration of the peptide in the bound state, but also on restrictions of the mobility, i.e., on the correlation time τc, which can be different for every residue; (2) the binding capacity of the synthetic peptides to mAbs measured by radioimmunoassay is directly correlated to the NOE magnitude; and (3) the combined interpretation of the COSY and TR‐NOESY experiments gives a qualitative information about the nature and the overall conformation of the sequence which is in contact with the mAb binding site.
Time‐Resolved study of network self‐organization from a biopolymeric solution
Authors: Emanuele A. , Di Stefano L. , Giacomazza D. , Trapanese M. , Palma‐Vittorelli M. B. , Palma M. U.
Published: December 2004

Time‐resolved studies of network self‐organization from homogeneous solutions of the representative biostructural polymer agarose are presented. Solutions are temperature quenched and observed by several techniques. Consistent with previous suggestions by the authors, experiments at concentrations up to about 1.75% w/v provide direct kinetic evidence for the occurrence of at least two distinct processes, leading, in sequence, to self‐assembly. These are as follows: (a) a liquid–liquid phase separation of the solution occurring via spinodal demixing and resulting in two sets of regions that have, respectively, higher and lower than average concentrations of random‐coiled polymers; and (b) the subsequent 2 coils → double helix transition and accompanying cross‐linking and gelation (due to branching of double helices), occurring in the high‐concentration regions. The size of the high‐concentration regions depends upon agarose concentration and quenching temperature, and is in the range from a fraction of micrometers to a few micrometers, in agreement with earlier experiments. Bundling of the double‐helical segments is known to follow self‐assembly and can be considered as a third step (gel curing). This follows from the thermo‐dynamic instability of the helical segments in the solvent, behaving as a system of rod‐like particles connected by more or less flexible joints.

The two processes leading in succession to self‐assembly are discussed in terms of a phase diagram consistent with available data. Their time scales differ remarkably. At the end of the first process, all polymers remain random coiled and freely drifting. Much later coil–helix transition is observed, always in coincidence with polymer cross‐linking and gelation. The enhancement of concentration of random‐coiled polymers in specific regions of the sol caused by spinodal demixing is thus a prerequisite for self‐assembly of these biostructural gels in the concentration interval studied. Conceptually, concentration enhancements of this type can provide a new pathway for promotion of functional biomolecular interactions even at very low average concentrations. The mechanism will work identically if the region of instability is reached by varying the polymer concentration (e.g., by biosynthesis), rather than by temperature quenching.
Evidence of three distinct conformations—single chain, single helix, and triple helix—of (1 → 3)‐β‐D‐xylan in the solid and intact frond of green algae as studied by 13C‐nmr spectroscopy
Authors: Saitô Hazime , Yamada Junko , Yoshioka Yuko , Shibata Yûkô , Erata Tomoki
Published: December 2004

High‐resolution solid‐state 13C‐nmr spectra of (1 → 3) ‐β‐D‐xylan from Bryopsis maxima and Caulerpa brachypus were recorded for various preparations from intact through dried frond to anhydrous and hydrated xylan, to gain insight into their secondary structures as compared with those of (1 → 3)‐β‐D‐glucans. It turned out that 13C‐nmr peaks of intact or dried frond were mainly ascribed to the peaks from (1 → 3)‐β‐D‐linked xylose residue, although some peaks were seen at the carbonyl and aliphatic regions. Dehydration of frond by either air drying or ethanol, and extraction of xylan with acid, did not result in substantial spectral change, although extraction with saturated ZnCl2 solution caused appreciable displacements of the C‐3 as well as C‐2 and C‐5 peaks. This means that no major conformational change was induced during the above‐mentioned processes of the dehydration and the extraction with acid. The extraction with ZnCl2 solution, however, induced major conformational change. Thus, we found that the above‐mentioned three distinct conformations–triple helix, single helix, and single chain–are also present in (1 → 3)‐β‐D‐xylan, as judged from the conformation‐dependent displacements of 13C chemical shifts, hydration/dehydration‐induced spectral change, and the x‐ray diffraction data on Penicillus dumetosus by Atkins et al. In particular, we found that all the preparations except for the extraction with ZnCl2 solution retain the triple helix form, whereas the anhydrous and hydrate samples of the latter preparations take the single‐chain and single‐helix form, respectively. Further, we measured the 13C spin‐lattice relaxation times of laboratory frame of the xylan to gain insight into better understanding of the role of the hydroxymethyl group as an efficient relaxation pathway in (1 → 3)‐β‐D‐glucan.
Molecular and crystal structures of Aib‐containing oligopeptides Boc‐Leu4‐Aib‐Leu4‐OBzl and Boc‐(Leu4‐Aib)2‐OBzl
Authors: Okuyama K. , Saga Y. , Nakayama M. , Narita M.
Published: December 2004

Sample peptides Boc‐Leu4‐Aib‐Leu4‐OBzl and Boc‐(Leu4‐Aib)2‐OBzl, were crystallized by the solvent‐evaporation method. Both crystals are monoclinic, with space group of P21 and Z = 2. The cell parameters are a = 16.580(7), b = 21.105(7), c = 11.583(4) Å, and β = 104.90(3)° (Boc‐Leu4‐Aib‐Leu4‐OBzl), and a = 15.247(9), b = 19.04(l), c = 16.311(9) Å, and β = 117.10(1)° [Boc‐(Leu4‐Aib)2‐OBzl]. Crystal structures were solved by the direct method and refined to R values of 0.096 (the former peptide) and 0.112 (the latter). Peptide backbones fold into a right‐handed α‐helix, except for the C‐terminal Aib residue in Boc‐(Leu4‐Aib)2‐OBzl. Both peptide molecules are stabilized by six (the former) or seven (the latter) intramolecular (5 → 1) hydrogen bonds, and arranged in the head‐to‐tail fashion, which makes an infinite column. In this column, one (the former) or two (the latter) intermolecular hydrogen bonds link the neighboring molecules. In the case of Boc‐Leu4‐Aib‐Leu4‐OBzl, the solvent molecule N, N‐dimethylformamide was found in the difference Fourier map. There was a hydrogen bond between peptide and solvent molecule. Along the lateral direction, only hydrophobic contacts were observed between adjacent peptide molecules.
Ionization of clusters. IV. Cooperativity and allosterism derived from shared clusters in macromolecules
Authors: Saroff Harry A.
Published: December 2004

Clusters of ionizable groups are examined for conditions that develop cooperativity (1) on the binding of protons, and (2) on the binding of an associated ligand when the clusters are shared between domains or subunits in macromolecules. Cooperative binding isotherms for protons have long been observed (but not emphasized as cooperative binding) when studies have been done on clusters for the evaluation of metal ion complexation [A. E. Martell & M. Calvin (1952) Chemistry of the Metal Chelate Compounds, Prentice‐Hall, Englewood Cliffs, New Jersey]. Reactions are formulated in this paper to show that anions, chelating to positively charged clusters, are also capable of developing the cooperative binding of protons. Extension of these simple reactions to those where clusters of ionizable groups are shared between domains of macromolecules provides models for cooperative binding, which include the allosteric, Bohr, anion, and cation effects in proteins.