bluuebunny/crossref_metadata_2025 huggingface dataset:
DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization
Authors: Ye Yun‐hua , Li Haitao , Jiang Xiaohui
Published: December 2004

3‐(Diethoxyphosphoryloxy)‐1, 2, 3‐benzotriazin‐4(3H)‐one (DEPBT) is an effective coupling reagent for synthesis of linear and cyclic peptides by both solution and solid‐phase peptide synthesis. DEPBT mediates amide bond formation with remarkable resistance to racemization. When DEPBT is used as a coupling reagent, it is not necessary to protect the hydroxy group of the amino component (such as tyrosine, serine, and threonine) and the imidazole group in the case of histidine. The high efficiency of DEPBT and its utility have been demonstrated in the syntheses of complex natural products such as ramoplanin A2, ramoplanose aglycon, ustiloxin, and teicoplanin aglycon. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005
Theory of the helix–coil transition in DNA considered as a copolymer
Authors: Lifson Shneior
Published: December 2004

The grand‐partition‐function theory of the preceding paper is used to derive expressions for the number of base pairs bonded in DNA with explicit consideration of the copolymeric nature of the DNA. The following features of the DNA molecule are taken into account: (1) the DNA's from different sources have different ratios of a–t and g–c pairs; (2) the a–t pair is weaker than the g–c pair; (3) the nearest‐neighbor frequencies of the two kinds of base pairs are nonrandom; (4) the stacking energies (nearest‐neighbor energies) of the various combinations of pairs may be different. An ensemble is constructed in which the features (1) through (4) are introduced by means of statistical weights on the various pairs and combinations of pairs, and an expression for the corresponding partition function is written. Expressions are derived for the number of bonded base pairs and the number of helical sequences.
Absorption and rotation of light by helical oligomers: The nearest neighbor approximation
Authors: Bradley D. F. , Tinoco I. , Woody R. W.
Published: December 2004

Explicit expressions for the DK and RK associated with the excitation of regular oligomers from their ground states to the Kth exciton state as functions of N, P, μ, μ∥, μt, and μr have been derived on the assumption that the only perturbation Vij to the N singly excited states results from exciton transfer between adjacent residues, V12. The DK relation predicts the occurrence of a perpendicularly polarized absorption band arising from μ⟂ and a parallel polarized band from μ∥ with maxima at K = [2(N + 1)/P] and 1, which are shifted from the monomer band by 2V12 cos(2π/P) and 2V12cos[π/(N + 1)], respectively. Overlapping of the bands, which may occur in oligomers, decreases rapidly with N, leaving two perfectly polarized bands in the polymer. Within each band which includes only a few levels near the maxima, the most probable transition may be associated with all of the intensity for very short chains but only 40.2–50% (⟂) and 80.9% (∥) as N → ∞ (polymer case). A relation was derived by which V12 may be simply calculated from μ, μr, μt, μ∥, a, and z. The nearest neighbor theory was compared with experiment by calculating a reasonable set of the relevant parameters which exactly account for known spectral shifts.
Polynucleotides. VI. Molecular properties and conformation of polyribouridylic acid
Authors: Richards Edward G. , Flessel C. Peter , Fresco Jacques R.
Published: December 2004

Optical, hydrodynamic, and titration properties of polyribouridylic acid (poly U) were investigated in 0.2M Na+, pH 6. Above 15°C., poly U was shown to be a randomly coiled single chain, devoid of secondary structure. There is no shear rate dependence of viscosity; molecular weight is independent of ionic strength; there is virtually no conformationally dependent hypochromicity; no anomalous optical rotatory dispersion; and no anomalous titration behavior. The dependence of the sedimentation constant and intrinsic viscosity on molecular weight was determined. Poly U was shown to be the most highly expanded of all synthetic and naturally occurring randomly coiled polynucleotides.
Studies of the conformation of amylose in solution
Authors: Rao V. S. R. , Foster Joseph F.
Published: December 2004

It has been suggested previously that water is a nearly indifferent solvent for amylose on the basis that the exponent a of the Mark‐Houwink equation is very nearly 0.50 and the second virial coefficient A2 is extremely small. It is shown that such solutions do not show the expected precipitation on cooling and the temperature coefficient of the intrinsic viscosity is small and negative. It is suggested that water is both an indifferent and a nearly athermal solvent for this polymer. Various lines of evidence suggest the polymer to exist as a very stiff coil in this and in other solvents. Variations in the intrinsic viscosity and radius of gyration in various solvents show the importance of skeletal effects in addition to the usual osmotic effects. Application of the Porod‐Kratky wormlike coil model leads to unreasonably short persistence lengths if the contour length is based on a fully extended conformation but large and reasonable values of the order 40–70 A. if it is assumed that the basic conformation is the helical one observed in various amylose complexes in the crystalline state. As was previously known, the specific optical rotation decreases sharply in aqueous solutions above approximately pH 12. Accompanying this change in rotation there is first a decrease, then an increase in intrinsic viscosity. This later behavior is strikingly reminiscent of the known behavior of polyglutamic acid in the pH‐dependent transition from helix to coil. It is concluded that the conformation is that of a stiff coil with essentially helical backbone contour in neutral solution, and the decrease in rotation and in intrinsic viscosity at high pH results from a partial breakdown of the helical structure with a concomitant increase in backbone flexibility. The increase in viscosity and radius of gyration in extreme alkaline solution is probably due to a polyelectrolyte expansion of the polymer coil. Some other miscellaneous observations which are in accord with this model are also pointed out.
Phonon dispersion curves and normal coordinate analysis of α‐poly‐L‐alanine
Authors: Fanconi Bruno , Small Enoch W. , Peticolas W. L.
Published: December 2004

The vibrational frequencies of α‐helical poly‐L‐alanine and its N‐deuterated analog have been assigned by normal coordinate analyses. The phonon dispersion curves and frequency distribution of α‐poly‐L‐alanine have been calculated by using a model which includes hydrogen bonding. The frequency distribution was used to interpret the inelastic neutron scattering data and to calculate the heat capacity. The low‐frequency chain modes involving accordian‐like motions of the whole helix have been calculated and their dispersion investigated by means of a simplified model.
Time dependent model for hemoglobin and allosteric enzymes. II.
Authors: Bush Robert Ter , Thompson Colin J.
Published: December 2004

The time‐dependent theory developed in Part I is specialized to treat tetrameric hemoglobin, and the results of the theory for dimeric‐and tetrameric hemoglobin are compared with data on the kinetics of the reactions of hemoglobin with carbon monoxide and oxygen at various salt concentrations for the case of large concentration of ligand relative to that of hemoglobin. The fit of the theoretical results to the data suggests that hemoglobin at a 2 M salt concentration is predominantly dimeric and that the tetramer should be taken as the functional unit to explain the kinetics of the reactions of normal hemoglobin. A relationship is established between the time‐dependent theory arid Adair's Intermediate Compound Hypothesis (I.C.H.) for hemoglobin, as brought to its present state by Gibson and Roughton. A generalization (G.I.C.H.) of the I.C.H. is presented and is shown to be equivalent to the time‐dependent theory in the limit of infinite ligand concentration. The I.C.H. is shown to be an excellent approximation to the centralized theory (G.I.C.H.) in this limit.
Proton transfer and polarizability of hydrogen bonds formed between cysteine and lysine residues
Authors: Kristof Wolfgang , Zundel Georg
Published: December 2004

(L‐Cys)n + N‐base systems and (L‐Cys)n + (L‐Lys)n systems were studied by ir spectroscopy. It is shown that in the water‐free systems, SH ⃛N ⇌ S− ⃛H+N hydrogen bonds are formed. With the (L‐Cys)n + N‐base systems, both proton‐limiting structures in the SH ⃛N ⇌ S− ⃛H+N bonds have equal weight when the pKa of the protonated N‐base is 2 pKa units larger than that of (L‐Cys)n. The same is true with the water‐free (L‐Cys)n + (L‐Lys)n system. Thus, with regard to the type of proton potentials present, these hydrogen bonds are proton‐transfer hydrogen bonds showing very large proton polarizabilities. This is confirmed by the occurrence of continua in the ir spectra. Small amounts of water open these hydrogen bonds and increase the transfer of the proton to (L‐Lys)n. In the (L‐Lys)n + N‐base systems, with increasing proton transfer the backbone of (L‐Cys)n changes from antiparallel β‐structure to coil. In (L‐Cys)n + (L‐Lys)n, the conformation is determined by the (L‐Lys)n conformation and changes depending on the chain length of (L‐Lys)n. Finally, the reactivity increase in the active center of fatty acid synthetase, which should be caused by the shift of a proton, is discussed on the basis of the great proton polarizability of the cysteine–lysine hydrogen bonds.
Intramolecular low‐frequency vibrations in lysozyme by neutron time‐of‐flight spectroscopy
Authors: Bartunik H. D. , Jollès P. , Berthou J. , Dianoux A. J.
Published: December 2004

High‐resolution neutron time‐of‐flight (TOF) spectra were measured for partly deuterated hen egg‐white lysozyme in solution with and without N‐acetyl‐glucosamine inhibitor bound to the cleft, and in polycrystalline form. Weak but reproducible bands occur at frequencies between 25 and 375 cm−1. The bands were tentatively assigned on the basis of previous results for homopolypeptides. At very small energy transfers (between about 1 cm−1 and 40 cm−1), the TOF spectra show a dependence both on inhibitor binding and crystalline environment. This is interpreted in terms of conformational flexibility.
Influence of ionic strength on the dichroism properties of polynucleosomal fibers
Authors: Lee Keun Su , Crothers Donald M.
Published: December 2004

We report electric‐dichroism and electron‐microscopic studies of chromatin fibers fixed by protein–protein crosslinking at salt concentrations ranging from 10 to 100 mM. The results confirm a progressive disorganization of the fiber as the salt concentration is lowered. The positive dichroism and large polarizability anisotropy characteristic of the 300‐Å diameter fiber found in 100 mM salt are replaced by negative dichroism and smaller effective polarizability anisotropy or dipole moment for samples fixed at lower salt concentration. We interpret the results in terms of segmental, field‐induced orientation of the disorganized structure which is present in low salt concentrations. We also observed a field‐induced absorbance decrease in chromatin fibers fixed at salt concentration at and below 100 mM. All three optical effects, namely overall orientation of the high‐salt fixed fiber, segmental orientation of the low‐salt fixed fiber, and field‐induced absorbance decrease, occur on roughly the same time scale, 20–100 μs for 50 nucleosome polynucleosomes. The polarizability anisotropy of fibers fixed in 100 mM salt was found to be proportional to the length of the fragment and to the reciprocal square root of the conductivity of the solution used for electric‐dichroism measurements. Addition of Mg2+ to the measurement buffer affected the dichroism amplitude of samples fixed below 100 mM salt but not those fixed at 100 mM salt. The results reinforce the need for caution in interpreting electric‐dichroism measurements on chromatin fibers because of possible field‐induced distortion effects.
Structure of the deoxytetranucleotide d‐pApTpApT and a sequence‐dependent model for poly(dA‐dT)
Authors: Viswamitra M. A. , Shakked Zippora , Jones P. G. , Sheldrick G. M. , Salisbury S. A. , Kennard Olga
Published: December 2004

The x‐ray structure of the hydrated ammonium salt of the deoxytetranucleotide d‐pApTpApT was determined by Patterson and direct methods at a resolution of 1 Å. The crystal structure contains right‐handed double‐helical segments formed by complementary Watson‐Crick‐type hydrogen bonding between the adenine and thymine bases of neighboring molecules. The minihelix contains two base pairs. The chains are antiparallel. The A‐T and T‐A sequences have different phosphodiester conformations. The deoxyribose‐pucker and the sugar–base orientation alternate along the chain depending on the nature of the base (3′‐endo for purine and 2′‐endo for pyrimidine). The extended structure is stabilized by base–base, base–sugar, and hydrogen‐bond interactions. The minihelix of two base pairs provides starting coordinates for model‐building studies of the dA‐dT polymer. A B‐DNA‐type polymer structure is described, which has sequence‐dependent alternations of both the deoxyribose pucker and the phosphate diester bridge conformation. Such sequence‐dependent DNA structures, if present locally in regions such as operator sequences, could facilitate sequence‐specific interactions. The crystal study also suggests possible geometrical parameters for the replication fork.
The chiroptical properties of proteins. III. Adenylate kinase
Authors: Snyder Robert W. , Hooker Thomas M.
Published: December 2004

The CD spectrum of the enzyme adenylate kinase has been investigated. Theoretical calculations, based on the x‐ray crystal structure, have been carried out by means of an origin independent matrix formalism. The entire molecule was included in the calculations in the sense that essentially all electronic transitions that occur at wavelengths longer than 185 nm were included in the basis set. A linear dielectric function was utilized to evaluate the intertransition coupling potentials. The results of the theoretical calculations were in reasonable agreement with experimental CD spectra of the molecule.
Dynamics of folding and unfolding transition in a globular protein studied by time correlation functions from computer simulation
Authors: Kanô Fumiaki , Gō Nobuhuro
Published: December 2004

A method of calculating time correlation functions from records of computer simulated equilibrium conformational fluctuations in a globular protein is discussed. Use of the calculated time correlation function for discussions of dynamics of folding and unfolding transition in the two‐dimensional lattice model of proteins. The time correlation functions can be approximated in general by a sum of two simple exponential terms. The relaxation time of the slower mode does not depend on the nature of the physical quantity with respect to which the time correlation function is calculated. This time characterizes the overall folding and unfolding transition. The relaxation time of the faster mode depends on the nature of the physical quantity and characterizes conformational fluctuations within each of the native and denatured states. The mechanism of a previously observed phenomenon of the acceleration of the folding and unfolding transition by short‐range interactions is discussed.
Visualization of the nature of protein folding by a study of a distance constraint approach in two‐dimensional models
Authors: Wako Hiroshi , Scheraga Harold A.
Published: December 2004

A distance constraint approach is applied to two‐dimensional models of proteins in order to visualize the nature of protein folding and to examine the relative roles of different ranges of interaction. Three different native structures (I, II, and III) are considered; they have two different kinds of residues, viz., hydrophobic and hydrophilic, and different sequences of these residues. We examine how the distance constraint approach functions in the prediction of protein folding when we know the sequence of the residues, the (fixed) bond lengths, the mean distances between residues i and i + 2, and i and i + 3, and the mean distances for hydrophobic–hydrophobic, hydrophobic–hydrophilic, and hydrophilic–hydrophilic contacts between residues i and i + j, where j ≥ 4. This approach involves optimization of an object function with respect to 98 variables and is not free of the multiple‐minimum problem. The optimization is always terminated if the chain is entangled and/or the segments (residues) are packed too compactly to move. In order to escape from such situations and to take the excluded‐volume effect into account, a Monte Carlo method is used after the optimization is trapped in local minima. Success in the prediction of folding is found to depend on the starting conformations and on the native conformations. Fair success is obtained in predicting the helix‐like structure in protein I and the overall structure of protein III, but not the β‐like structures of proteins I and II. Insofar as the prediction of the structure of protein III is reasonable, it appears that some sequences of residues produce greater constraints on their conformations than others, if one considers only the hydrophobic and hydrophilic nature of the residues. These results imply that, in the folding of real proteins in three dimensions, the competition for hydrophobic (and hydrophilic) residues for inside (outside) positions in the molecule probably constitutes a necessary but not a sufficient condition to form and stabilize the native structure. The failure to predict the structure of protein II, and part of that of protein I, suggests that there are two types of long‐range interactions. One (which we considered here) is nonspecific (i.e., is defined only in terms of contacts between residues of the same or different polarity) and acts at any stage of protein folding; the other (which we did not consider here) is a specific interaction between residues in pairs and contributes only when the residues in the specific pair take on the native conformation. Presumably, incorporation of such specific long‐range interactions, together with the nonspecific ones, is necessary for successful protein folding, using the distance constraint approach.
Interactions of molecules with nucleic acids. VI. Computer design of chromophoric intercalating agents
Authors: Miller Kenneth J. , Newlin Donald D.
Published: December 2004

The mode of action of many antitumor agents entails the inhibition of nucleic acid synthesis. Because many of the drugs can intercalate, it is assumed that intercalation is an important step in the mechanism of biological activity. As intercalants contain a planar chromophore as an ingredient essential for intercalation, chromophores that should fit into DNA are desired. This is the main theme of this investigation. Binding to DNA of fundamental moieties, protonated pyridine, aniline, phenol, quinone, and 4H‐thiopyran‐4‐one, is studied to determine their optimum placement in DNA. The optimum orientations for each moiety are superimposed to form polyaromatic systems that can intercalate in a manner in which functional groups on these chromophores are oriented as in the moieties themselves. Ideal intercalants proposed contain three and four fused ring system, have protonated ring nitrogen atoms located to maximize the electrostatic interactions with DNA, hydroxy and amino groups that can hydrogen bond to the OII and O5′ phosphate backbone atoms, and carbonyl and sulfur groups in the central position of the ring system to provide variations in the chromophore and to interact with the relatively positive region in the intercalation site. The optimum orientation occurs when the chromophore and the base pairs overlap to the maximum extent. The ideal intercalants are fundamentally of the type: magnified image
25Mg‐nmr investigations of the magnesium ion–DNA interaction
Authors: Rose D. Murk , Polnaszek C. F. , Bryant R. G.
Published: December 2004

25Mg‐nmr data are reported that address the nature of the magnesium ion–DNA interaction. It is found that competitor ions such as calcium, mercury, zinc, and cobalt ions are not effective in competing for all of the magnesium ion–DNA interaction that is reported by the 25Mg‐nmr spectrum. The temperature dependence of the 25Mg‐nmr spectrum in DNA solution studied at high concentrations of competitor ion indicates that the chemical‐exchange lifetime of the magnesium ions at DNA binding sites makes a major contribution to the 25Mg‐nmr line width. However, the activation parameters are not consistent with the temperature dependence of either transport properties or chemical exchange with phosphate groups alone, but are consistent with a sum of at least two processes that provide opposing contributions to the 25Mg‐nmr relaxation. It is also shown that the non‐Lorentzian character of the 25Mg‐nmr line previously reported is consistent with the effect of an incompletely averaged static nuclear electric quadrupole interaction and/or an exchange process that is slow with respect to the magnitude of this interaction. Because the concentrations employed in these experiments are high, the present data do not provide a direct or critical test of the electrostatic theories of ion–polyelectrolyte interaction. The present data do demonstrate, however, that such theories alone are insufficient as a basis for understanding the 25Mg‐nmr data.
Stable cruciform formation at inverted repeat sequences in supercoiled DNA
Authors: Benham Craig J.
Published: December 2004

This paper analyzes equilibrium superhelical cruciform formation in a topologial domain of DNA containing inverted repeat sequences. The cruciform conformation is shown to be stable when the molecule is sufficiently negatively supercoiled but not when it is positively supercoiled. For a particular sequence containing a single inverted repeat, onset of stability occurs at a degree of negative superhelicity that depends critically on the number of base pairs separating the repeat copies. The free energy associated with the stable cruciform state is constant, independent of the degree of superhelicity, up to the point where the complete inverted repeat participates in the cruciform. In contrast, the free energy of the alternative, unextruded state grows approximately quadratically with the superhelical deformation. Therefore, a degree of negative supercoiling occurs at which the cruciform state becomes energetically favored. The equilibrium properties of this cruciform extrusion transition vary with segment length, the positions and sizes of the inverted repeats involved, environmental conditions, and the degree of superhelicity imposed. In a segment containing multiple repeats, both tandem and inverted, of one or more sequences, the pattern of cruciforms that form is dependent on superhelicity in a complex way. Specific cruciforms may occur at equilibrium only in narrow ranges of superhelicity, their reabsorption being coupled to the extrusion of others.
Stereospecific oxidation of ascorbic acid by chiral association complexes between polypeptides and iron(III) chelate ions
Authors: Barteri M. , Pispisa B.
Published: December 2004

2,2,′,2″,2‴‐Tetrapyridineiron(III) complex ions anchored to poly(L‐glutamate) (FeL) or poly(D‐glutamate) (FeD) were used as catalysts for the H2O2 oxidation of L(+)‐ascorbic acid at pH 7 and varying complex:polymer‐residue molar ratios [C]/[P]. Evidence is produced that the reaction is a composite process reflecting contributions from parallel pathways, one of which corresponds to a catalytic route and is [H2O2]‐independent and the other to an uncatalyzed electron‐transfer process between the ascorbate anion and hydrogen peroxide. Stereospecific effects in the catalysis are observed on increasing the complex:polymer ratio, which corresponds to an increase of the amount of α‐helical fraction in the polypeptide supports (xa). Thus, at [C]/[P] = 0.01 (xa < 0.05), kFeD/kFeL = 1.0; but at [C]/[P] = 0.20 (xa ≈ 0.70), kFeD/kFeL = 4.0 ± 0.5, kFeD and kFeL being the second‐order rate constants of the electron‐transfer reaction between the FeD or FeL isomer of the asymmetric catalyst and the L‐ascorbate anion. The activation energies were found to increase markedly on going from the former to the latter complex:polymer ratio but, at the same time, to exhibit equal values with both enantiomeric catalysts. Stereoselectivity therefore appears to be an entropy‐controlled process, arising from the conformational rigidity of the precursor complex, which very likely sees the substrate molecules bound to the chiral residues of the ordered polymer surrounding the active sites. The implications of the stereochemical features of the substrate–catalyst adduct on the mechanism of electron transfer are also discussed. Evidence is presented that the asymmetric [Fe(tetpy)(OH)2]+–polyelectrolyte systems play the additional role of environmental controller of the uncatalyzed oxidation of the L‐ascorbate anion.
Effects of pressure on the composition density distribution in the ultracentrifuge
Authors: Sharp Dan S. , Ifft James B.
Published: December 2004

The effects of pressure on the composition density distribution in a binary density gradient at sedimentation equilibrium in the analytical ultracentrifuge are rigorously examined. A computer algorithm is described for the necessary iterative computations. The pressure effect is found to be significant in runs where a long column length, high angular velocity, and a salt with a high pressure correction coefficient are simultaneously employed. Such conditions are sometimes encountered in current studies in which high precision is required to measure the compressibility of proteins.
Quasielastic light scattering by biopolymers. VI. Diffusion of mononucleosomes and oligonucleosomes in the presence of static and sinusoidal electric fields
Authors: Schmitz Kenneth S.
Published: December 2004

Quasielastic light scattering and electrophoretic light scattering studies were carried out on mononucleosome and oligonucleosome systems. The electrophoretic light scattering experiments employed static and sinusoidal electric fields. Data are presented that suggest at least two relaxation modes. It is proposed that the small amplitude sinusoidal field effectively polarizes the ion atmosphere about the polyion, thus leading to an induced dipole moment that varies sinusoidally in time. This model is, in essence, an extension of the current interpretation of low‐frequency dielectric dispersion data on DNA as being due to fluctuations of counterions along the polyion.
15N‐NMR spectroscopy. 34. Stereospecificity of the polymerization of D,L‐leucine N‐carboxyanhydride and γ‐OMe‐D,L‐glutamic acid N‐carboxyanhydride
Authors: Kricheldorf H. R. , Hull William E.
Published: December 2004

D,L‐Leucine‐N‐carboxyanhydride (D,L‐Leu‐NCA) and γ‐methyl‐D,L‐glutamic acid N‐carboxyanhydride (γ‐OMe‐D,L‐Glu‐NCA) were synthesized with ca. 2.5% 15N enrichment. Their polymerizations were conducted under a variety of conditions using benzylamine, triethylamine potassium tert‐butanolate, and pyridine as initiators. The 40.55‐MHz 15N‐nmr spectra of the resulting stereocopolypeptides measured in trifluoroacetic acid display at least four signals, representing the isotatic, syndiotactic, and two heterotactic triads. From the signal intensities it was concluded that these NCAs behave nearly identically. With benzylamine initiation the formation of isotactic blocks is slightly favored, and they are still more predominant when strong bases are used as initiators. Initiation by pyridine favors the formation of syndiotactic sequences. However, in all cases the average lengths of the stereoblocks never exceeded 4 monomer units. The low stereospecificity of most polymerizations of D,L‐NCAs is confirmed by the high degree of solubility of the resulting poly(D,L‐amino acids) in aprotic solvents. Penultimate effects are weak or absent, so that most polymerizations follow Bernoullian type statistics. Deviations from these statistics were found for polymerizations in pyridine.
Structure of methylene blue–DNA complexes studied by linear and circular dichroism spectroscopy
Authors: Nordén Bengt , Tjerneld Folke
Published: December 2004

Bradley et al. [(1972) Biopolymers 11, 645–652] used electro‐optical measurements to show that methylene blue (MB), like acridine orange, in its DNA complex is oriented more or less perpendicular to the helix axis as expected if intercalated. High‐precision flow linear dichroism (LD) here confirms that MB is coplanar with the DNA bases at low dye/DNA binding ratios and low ionic strengths. CD shows two origins of induced optical activity for the transition of lowest energy (polarized parallel to the long‐axis of the dye molecule): at low binding ratios (r < 0.05), a weak monomeric CD with the same shape as the absorption curve is observed, while at higher binding ratios, a strong exciton CD dominates due to interaction between pairs of MB ligands. The monomeric CD spectrum shows a remarkable dependence on ionic strength: it is negative in the absence of extra salt, but changes sign (at ca 20 mM NaCl or 0.15 mM MgCl2) and becomes essentially the positive mirror‐image spectrum at high ionic strengths (>300 mM Na+ or >0.4 mM Mg2+). Nondegenerate coupled‐oscillator theory can explain the CD in terms of interactions of transition moments of the dye and the nearest nucleotide bases and indicates a change between two intercalation geometries: a Lerman type of mode, denoted γ−, and an orthogonal mode, denoted γ+. This rotation of MB in the base‐pair pocket is accomplished at Na+ and Mg2+ concentrations when the phosphates are effectively screened and the result suggests a more localized bonding of Mg2+ than is expected from simple polyelectrolyte models. The exciton effect at high binding ratios, observed both in CD and in LD, can be interpreted in terms of an interaction between an intercalated and a nonintercalated MB. The geometry of this “accidental” dimer is consistent with a location of the nonintercalated MB in the minor groove, bridging the strands by the positively charged amino groups directed towards phosphate groups. The dihedral angle of the MB pairs, corresponding to a left‐handed helix, is opposite to that with acridine orange and proflavine on DNA, indicating that the latter ligands bind to DNA in a different way.
A circular dichroism microspectrophotometer
Authors: Maestre M. F. , Katz J. E.
Published: December 2004

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200‐ and 800‐nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.
Conformational properties of microtubule protein: Their relation to the self‐assembly process in vitro
Authors: Bayley Peter M. , Clark David C. , Martin Stephen R.
Published: December 2004

Near‐ and far‐uv CD spectra of microtubule protein preparations have been examined to study the possible role of protein conformation in relation to the kinetics of the self‐assembly of these proteins into microtubules in vitro. Although tubulin can form conformations with high helical content under apolar solution conditions, this transformation is apparently not involved in self‐assembly. There is no major perturbation of tubulin near‐uv CD by reagents and solution conditions favoring assembly. Thus, in these preparations, tubulin, as dimer and as oligomer with MAPs, is effectively in the conformation in which it undergoes self‐assembly. This conclusion is consistent with a hybrid model of assembly of microtubule protein involving direct incorporation of oligomeric species as an alternative to the condensation polymerization of tubulin dimer as the exclusive assembly mechanism.
Solid‐state geometry and conformation of linear, diastereoisomeric oligoprolines
Authors: Benedetti Ettore , Bavoso Alfonso , di Blasio Benedetto , Pavone Vincenzo , Pedone Carlo , Toniolo Claudio , Bonora Gian Maria
Published: December 2004

We have carried out a systematic analysis of the solid‐state conformational preferences of a number of linear homo‐oligoprolines (to the tetramer) by ir absorption and x‐ray diffraction. The peptides present different chiral sequences (tacticities), various types (urethane and amide) of N‐protecting groups, and free and blocked C‐termini (which imply different capabilities of forming H‐bonds). The following conclusions can be drawn: (i) values for the geometry of the prolyl residue and the peptide bond in the cis and in the trans conformations are proposed; (ii) in general the conformational angles φ and ψ in the linear homo‐oligoprolines have values appropriate for the polyproline II structure (conformation F); (iii) the pyrrolidine ring shows various types of puckering with no apparent relation to the backbone conformation; (iv) Pro‐Pro peptide bonds generally take the trans conformation, the few cases of cis conformation being formed by Pro residues of different chirality; (v) the single H‐bond donor — OH, when present, is always bonded to H‐acceptors, which can be either the urethane or the amide or the peptide carbonyl but never the carbonyl group of the — COOH moiety.
Electron diffraction studies of poly(α‐aminoisobutyric acid)
Authors: Malcolm B. R.
Published: December 2004

Electron diffraction photographs of poly (α‐aminoisobutyric acid) treated with dichloracetic acid are shown to be consistent with space group R3c. This is strong evidence for a hexagonal cell in which right‐ and left‐handed 310‐helices form a honeycomb structure. It appears that the dichloracetic acid molecules are located in the centers of the holes.
X‐ray diffraction study of the interaction of the amino terminal half‐molecule of histone H4 with duplex DNA
Authors: Wachtel E. J. , Sperling R.
Published: December 2004

The interaction of the amino terminal half‐molecule of histone H4 with duplex DNA has been studied by fiber x‐ray diffraction. Changes induced in the diffraction pattern of B‐DNA by the presence of the bound peptide have been Fourier‐analyzed and the results presented in terms of a deweighted radial projection of the electron density. We conclude that the peptide binds on the major groove side of the sugar–phosphate chain.
Serine and β‐turns
Authors: Aubry A. , Marraud M.
Published: December 2004

Serine is one of the most frequent residues included within β‐turns in proteins. Conformational investigations have been carried out on the LL and LD model dipeptides ButCO‐Pro‐Ser‐NHMe in the solute state by ir and nmr spectroscopies and in the solid state by x‐ray diffraction. LL and LD sequences are folded in the βI‐ and βII‐types, respectively, and in both cases a weak intramolecular interaction takes place between the NH and Oγ sites of the serine residue.
Neurophysin–neuropeptide hormone complexes: Biosynthetic origin and noncovalent interactions
Authors: Chaiken Irwin M.
Published: December 2004

Recent studies of the neurophysins and associated neuropeptide hormones have addressed both the biosynthetic pathways by which these noncovalent protein–peptide complexes are derived in neurosecretory neurons and the nature of the noncovalent interactions likely to occur during transport and storage in neurosecretory granules within the neurons. In vitro translation of hypothalamic mRNA and sequencing of cDNA obtained from this mRNA have yielded chemical evidence that each complex of hormone and major neurophysin is made through a common precursor molecule. The mature complexes obtained upon proteolytic processing of precursors exhibit interdependent hormone binding and self‐association interactions. Photoaffinity labeling and quantitative affinity chromatography have helped detect and define the binding surfaces involved. Further study of the structural nature of these surfaces is being carried out using large neurophysin fragments obtained by limited tryptic proteolysis.
Photo‐CIDNP study of interactions of serine proteinases with their protein inhibitors
Authors: Muszkat K. A. , Weinstein S. , Khait I. , Vered M.
Published: December 2004

Photochemically induced dynamic nuclear polarization was used to study the accessibility of surface tyrosine and tryptophan residues in proteinases, in their protein inhibitors and in the proteinase–inhibitor complexes. The accessibility probe is the triplet of 10‐(carboxyethyl) flavin formed by optical excitation. On complex formation we observe accessibility loss in the surface tyrosines and tryptophans in the proximity of the proteinase–inhibitor contact site, and in the case of bovine pancreatic trypsin inhibitor, in more distant tyrosines as well.
Conformational and dynamic considerations in the design of peptide hormone analogs
Authors: Hruby Victor J. , Mosberg Henry I. , Sawyer Tomi K. , Knittel James J. , Rockway Todd W. , Ormberg James , Darman Paul , Chan W. Y. , Hadley Mac E.
Published: December 2004

Efforts to understand the chemical‐physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical‐physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen1]‐oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure–activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α‐melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).
Synthesis and spectroscopic characterization of insulin derivatives containing one or two poly(ethylene oxide) chains at specific positions
Authors: Ehrat M. , Luisi P. L.
Published: December 2004

We describe a synthetic method aimed at specifically coupling poly(ethylene oxide) (PEO) to proteins without altering their spectroscopic properties. To do so, we have first modified the alcohol end group of PEO to an acid end group (PEO‐COOH). Coupling of PEO‐COOH to Gly A1 methylsulfonylethyloxycarbonyl (Msc) protected insulin yielded NαB1,NϵB29‐(PEO)2‐insulin. Conversely, coupling of PEO‐COOH to insulin whose Gly A1 and Lys B29 amino groups were protected with Msc yielded NαB1‐PEO‐insulin. The products were obtained in a spectroscopically pure form and characterized by uv and CD spectroscopy. Conformational and biological studies are in progress.
Methods for the analysis of the kinetics of cooperative binding of ligands to a two‐site lattice
Authors: Edgerton Mary E. , Handler Frank A. , Wu Cheng‐Wen
Published: December 2004

Solutions are obtained that describe the time dependence of the reversible binding of a ligand to a two‐site lattice. The binding may be cooperative. Three methods are used to obtain these solutions: the separation of on/off processes with a variable transformation, the asymptotic series analysis, and the singular perturbation procedure. Applications to parameter calculation from experimental data are presented. This kinetic system is such that it is difficult to extract all kinetic parameters from data analysis, and the implications for each method are also discussed.
Effects of composition and polymerization conditions on crosslinked polymer supports based on N‐acrylyl pyrrolidine and N,N‐dimethyl acrylamide for peptide synthesis
Authors: Varadarajan K. , Fiat D.
Published: December 2004

Poly(N‐acrylyl pyrrolidine‐co‐N,N′‐bis(acrylyl) 1,2‐diaminoethane‐co‐N‐Acrylyl 1,6‐diamino hexane HCl) with 2,5,8,11,12,15, and 20% degrees of crosslinking (DC) and poly‐(N,N′‐dimethylacrylaminde‐co‐N,N′‐bis(acrylyl) 1,2‐diaminoethane‐co‐N‐acrylyl 1,6‐diamino hexane HCl) with 2,5,10, and 15% DC have been synthesized in an entirely beaded form by reverse‐phase suspension polymerization. The monomer‐to‐water ratio was kept constant for all these polymers. With the exception of 2% crosslinked resins in the PAP and PDMA groups, all the resins exhibited favorable swelling properties in solvents normally used for peptide synthesis and the percent incorporation of the first amino acid residue was close to 100 for DC of up to 20%. This is explained as due to the flexible and polar characteristics of the crosslinking agent, N,N′‐bis(acrylyl) 1,2‐diaminoethane. Between 5 and 20% DC the degree of swelling of the resins changes only slightly with increasing DC. This effect of DC on the swelling and percent incorporation of the first amino acid residue is more favorable for peptide synthesis than in the case of divinyl benzene crosslinked poly(styrene).
Differences in structure and stability between normal and deuterated proteins (phycocyanin)
Authors: Chen Chang‐Hwei , Liu I‐Wen , MacColl Robert , Berns Donald S.
Published: December 2004

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔHd) and the temperature (td) of thermal denaturation of normal and deuterated phycocyanins isolated from two blue‐green algae, Plectonema calothricoides and Phormidium luridum. Values of td in deuterated proteins are about 5°C lower than those in normal proteins. The magnitudes of ΔHd in deuterated proteins are 18–36% lower than in normal proteins. The heatcapacity change (ΔCp) in protein unfolding is essentially the same (2 kcal/mol/K) for deuterated and normal proteins within the experimental error. At close to physiological temperature (27°C), the differences in thermodynamic functions in the native and denatured states are much higher in normal proteins than in deuterated proteins. CD was employed to evaluate both the secondary structures and urea denaturation of these two types of proteins. In P. luridum, deuterated protein is about 8% higher in α‐helix content; in P. calothricoides it is not significantly higher. Deuterated proteins are less resistant to the denaturant urea than are normal proteins: the denaturant concentration at the midpoint of the denaturation curve is 0.6–1.2 mol/L lower in the deuterated proteins. The apparent free energies of unfolding of deuterated proteins at zero denaturant concentration are 1.1–1.5 kcal/mol less than for normal proteins.
Salt‐dependent conformational transitions in the self‐complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation
Authors: Marky Luis A. , Blumenfeld Kenneth S. , Kozlowski Sharon , Breslauer Kenneth J.
Published: December 2004

Differential scanning calorimetry (DSC), temperature‐dependent uv‐absorption spectroscopy, and temperature‐dependent CD were used to monitor and characterize the salt‐dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex‐to‐single‐strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na+), a single, monophasic transition curve was observed. At lower salt (⩽0.01M Na+), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.
Geometry and mechanics of DNA superhelicity
Authors: Benham Craig J.
Published: December 2004

This paper analyzes the elastic equilibrium conformations of duplex DNA constrained by the constancy of its molecular linking number, Lk. The DNA is regarded as having the mechanical properties of a homogeneous, linearly elastic substance with symmetric cross section. Integral representations of the writhing number Wr and of Lk are developed, in terms of which the equilibria are given as solutions to an isoperimetric problem. It is shown that the Euler angles defining equilibrium conformations must obey equations identical to those governing unconstrained equilibria. A scaling law is developed stating that molecules supercoiled the same amount ΔLk will have geometrically similar elastic equilibria regardless of their length. Thus, comparisons among molecules of properties related to their large‐scale tertiary structure should be referred to differences in ΔLk rather than to their superhelix densities. Specific conditions on the elastic equilibrium conformations are developed that are necessary for ring closure. The equilibrium superhelical conformations accessible to closed‐ring molecules are shown to approximate toroidal helices. Questions relating to the stability and nonuniqueness of equilibria are treated briefly. A comparison is made between these toroidal conformations and interwound configurations, which are shown to be stable, although they are not equilibria in the present sense. It is suggested that entropic factors are responsible for favouring the toroidal conformation in solution.
X‐Ray diffraction studies on the structure of hydrated collagen
Authors: Sasaki Naoki , Shiwa Shinichi , Yagihara Shin , Hikichi Kunio
Published: December 2004

The temperature dependence of the humidity‐sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x‐ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.