bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectrophotometric and nmr analysis of the interaction of fluorinated mono‐ and bifunctional interacalators with DNA, poly(dA‐dT), and poly(dG‐dC)
Authors: Delbarre A. , Brown S. C. , James T. L. , Shafer R. H.
Published: December 2004

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA‐dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by 19F‐nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA‐dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG‐dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG‐dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left‐handed to a right‐handed conformation.
A lipid vesicle system for probing voltage‐dependent peptide–lipid interactions: Application to alamethicin channel formation
Authors: Woolley G. Andrew , Deber Charles M.
Published: December 2004

A membrance potential is shown to be established in phosphatidylcholine/cholesterol unilamellar vesicles using valinomycin in conjunction with a potassium ion gradient; this potential is monitored using the externally added fluorescent dye Safranine O. In the same system, transmembrance calcium fluxes are then detected using the (internally trapped) fluorescent dye Quin‐2. The calcium‐transport behavior of the channel‐forming peptide alamethicin is shown to be potential dependent in this system, in contrast to calcium transport by the ionophore Br‐A23187, which is unaffected by the potential. The observation of this potential‐dependent behavior for alamethicin suggests that this vesicle system may be suitable for direct spectroscopic observation of the voitage‐gating process.
Bicyclic peptides: Solution conformation and Ca2+ binding of the heterodetic bicyclic decapeptide cyclo(Glu1‐Leu2‐Pro3‐Gly4‐Ser5‐Ile6‐Pro7‐Ala8)‐cyclo(1γ → 5β) Phe9‐Gly10
Authors: Zanotti Giancarlo , Pinnen Francesco , Lucente Gino , Paolillo Livio , D'Auria Gabriella , Trivellone Enrico , D'Alagni Maria
Published: December 2004

The conformational behavior of a heterodetic bicyclic decapeptide (BCPLT) in the absence and in the presence of calcium ions has been studied by means of mono and two‐dimensional nmr techniques. Free BCPLT possesses a quite compact structure stabilized by intramolecular bonds and turns. In the structure a cluster of carbonyls is located in a cavity that is supposed to be the cation binding site.
Biologically significant conformation of the Saccharomyces cerevisiae α‐factor
Authors: Naider Fred , Jelicks Linda A. , Becker Jeffrey M. , Broido Michelle S.
Published: December 2004

The conformation of the tridecapeptide α‐factor of the yeast Saccharomyces cerevisiae was examined in both solution and in the presence of lipid vesicles. CD, differential scanning calorimetry, and phosphorus nmr all indicate that this mating pheromone interacts with lipid vesicles. In both aqueous and organic solution of the α‐factor is a flexible molecule that exhibits features of a type II β‐turn spanning the center of the peptide. Two‐dimensional Nuclear Overhauser enhancement spectroscopy gives evidence that the β‐turn is stabilized on interaction of the peptide with lipid vesicles. Our current belief is that the β‐turn may play an important role in the biologically active conformation of the α‐factor.
Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: Nuclear overhauser effect and energy minimization studies
Authors: Raghothama S. , Ramakrishnan C. , Balasubramanian D. , Balaram P.
Published: December 2004

The conformation of cyclolinopeptide A [cyclo(Pro–Pro–Phe–Phe–Leu–Ile–Ile–Leu–Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H‐nmr. A complete assignment of all CαH and NH resonances has been accomplished using two‐dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and J values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI β‐turn centered at Pro(1)‐Pro(2), with a cis peptide bond between these residues and a γ‐turn (C7) structure centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO—Phe(3)NH (4 → 1) and Leu(5) CO—Ile(7)NH (3 → 1) are observed in the low‐energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.
Low‐frequency dynamics and Raman scattering of crystals, of B‐, A‐, and Z‐DNA, and fibers of C‐DNA
Authors: Lamba Om. P. , Wang A. H.‐J. , Thomas George J.
Published: December 2004

Normal modes of vibration of DNA in the low‐frequency region (10–300 cm−1 interval) have been identified from Raman spectra of crystals of B‐DNA [d(CGCAAATTTGCG)], A‐DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z‐DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures—at 18 ± 2 cm−1 in the B‐DNA crystal, near 24 ± 2 cm−1 in A‐DNA crystals, and near 30 ± 2 cm−1 in Z‐DNA crystals—are shown to correlate well with the degree of DNA hydration in the crystals structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531–535; C. Demarco et al. (1985) Biopolymers 24, 2035–2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B‐, A‐, C‐, and Z‐DNA structures can be distinguished from one another on the basis of their characteristics Raman intensity profiles in the region of 40–140 cm−1, even through all structures display two rather similar and complex bands centered within the intervals of 66–72 and 90–120 cm−1. The similarity of Raman frequencies for B‐, A‐, C‐, and Z‐DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90–120 cm−1 in all double‐helix structures is due to in‐plane librational motions of the bases, which involve stretching of the purine‐pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G ⃛ C pairs (> 100 cm−1) than in structures containing A ⃛ T pairs (< 100 cm−1), consistent with the strengths of G ⃛ C and A ⃛ T hydrogen bonding.
Dynamic light scattering studies of porcine submaxillary mucin fractions in dilute solution at intermediate scattering vectors
Authors: Varma B. K. , Demers A. , Jamieson A. M. , Blackwell J. , Jentoft Neil
Published: December 2004

We report dynamic light scattering measurements over a wide range of scattering vectors for fractionated samples of porcine submaxillary mucin (PSM) glycoproteins in two different solvents: 0.1M NaCl, and 6M GdnHCl. The relaxation spectrum has been successfully resolved into a slow mode corresponding to pure translational diffusion and a fast mode containing information on the relaxation times for intramolecular motion. Analysis of the slow mode permits a light scattering evaluation of the polydispersity of these high molecular weight mucin glycoprotein fractions. Determination of the longest intramolecular relaxation times τ1 shows that these are much longer for the PSM fractions in 0.1M NaCl compared to 6M GdnHCl. These data are consistent with earlier studies showing that the chain conformation is the same in both solvents, but that in 0.1M NaCl, the PSM glycoprotein undergoes a self‐association process that is end‐to‐end in nature. Since the τ1 value is intimately related to the viscoelastic behavior of PSM solutions and gels, it is interesting to speculate that the end‐to‐end association process may be physiologically important.
Modeling DNA supercoils and knots with B‐spline functions
Authors: Hao Ming‐Hong , Olson Wilma K.
Published: December 2004

A method is offered to model the complex trajectories of closed circular DNA supercoils and knots. The trajectories are approximated by polygons and analytical expressions of the curves are generated from the polygons with B‐spline functions. The resulting curves are used to evaluate the writhe and elastic energy of a series of interrelated supercoils, and to generate detailed atomic models of the deformed double helix.
The 9‐fluorenylmethyloxycarbonyl group as a 5′‐OH protection in oligonucleotide synthesis
Authors: Ma Yunxi , Sonveaux Etienne
Published: December 2004

Oligo‐DNAs are synthesized on a solid support using the 9‐fluorenylmethyloxycarbonyl group as a 5′‐OH base labile protection. The synthesis of the pure protected nucleotides, a relevant phosphoramidite‐type strategy of coupling, and the optimization of the deprotection steps are described. This new synthetic method is an alternative to the standard protocol that avoids acidic conditions.
Structure of DNA hydration shells studied by Raman spectroscopy
Authors: Tao N. J. , Lindsay S. M. , Rupprecht A.
Published: December 2004

We have used Raman scattering to study the water O‐H stretching modes at ∼ 3450 and ∼ 3220 cm−1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220‐cm−1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen‐bond network of water is disrupted in the primary hydration shell (which therefore cannot have an “ice‐like” structure). The number of water molecules in the primary hydration shell was determined from the intensity of the ∼ 3200‐cm−1 band as about 30 water molecules per nucleotide pair. The ∼ 3400‐cm−1 O‐H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5–6 tightly bound water molecules per nucleotide pair remain. The frequency of the ∼ 3400‐cm−1 O‐H stretch mode is lower by 30 to 45 cm−1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the ∼ 3200‐cm−1 band and the shift of the ∼ 3400‐cm−1 O‐H stretch band provide a reliable way of measuring the hydration number of DNA.
Conformation of a bulge‐containing oligomer from a hot‐spot sequence by NMR and energy minimization
Authors: Woodson Sarah A. , Crothers Donald M.
Published: December 2004

Two‐dimensional nmr data on a bulge‐containing oligodeoxyribonucleotide, 5′dGATGGGCAG · dCTGACCCATC, and a regular oligomer of similar sequence, 5′dGATGGCAG · dCTGCCATC, are presented. The nonexchangeable protons are assigned from sequential nuclear Overhauser effect spectroscopy (NOESY) connectivities. The two‐dimensional NOE (NOESY) and correlated (COSY) spectra of the bulge‐containing oligomer are compared to those of the perfect 8‐mer. Experimental proton‐proton distances are determined from NOESY spectra acquired with mixing times of 100, 150, and 200 ms, using comparable distances in the B‐DNA region of the molecule as a calibration. With this approach, measured distances do not depend systematically on mixing time. Energy minimization techniques are used to calculate a three‐dimensional structure for the bulge‐containing oligomer in agreement with the nmr data. The helix is of the B family, with the extra adenine stacked into the helix, and the helix axis is bent by 20°.
Counterion diffusion in heparin solutions
Authors: Tivant P. , Perera A. , Turq P. , Belloni L.
Published: December 2004

The physiological importance of heparin is due to its strong interaction with bivalent counterions, especially Ca2+. A diffusional approach of this property is presented in this article: the observable is the self‐diffusion coefficient of the counterions, as a function of the ratio of the polyelectrolyte over the added salt concentrations. All the results are in agreement with a simple “quasi‐chemical model” in which two different states are assumed for the counterions: “free” or “bound.” The proportions of these two types of ions are calculated according to the distribution function of the counterions around the polyion. We assume that those of counterions located at a distance closer than a, the characteristic distance, are bound; the others are free. The ionic distribution function is evaluated by a numerical integration of a cell model Poisson–Boltzmann equation. Finally, this model leads to a very good agreement with the experimental results, if the radius of heparin polyion is assumed to be 6 and 10 Å.
Sequence dependence of the B‐A conformational transition of DNA
Authors: Mazur J. , Sarai A. , Jernigan R. L.
Published: December 2004

We have studied, by conformational analysis, the sequence dependence of DNA conformational transition between B‐ and A‐forms. We have considered intramolecular interactions between base pairs, without backbone, to examine their role in the conformational transition between B‐ and A‐forms, and found that base pairs themselves usually have intrinsic conformational preferences for the B‐ or A‐form. Calculation of all ten possible base steps shows that the base combinations, CC (or GG), GC, AT, and TA, have tendencies to assume the A‐conformation. Results show that it is particularly easy to slide along the long axis of the base pair for these steps, with AT and CC showing especially flat energies. These calculations show that a preference for the B‐ or A‐conformation depends on the electrostatic energy parameters, in particular, on dielectric and shielding constants; the A‐conformations are mainly stabilized by electrostatic interactions between favorably juxtaposed atomic charges on base pairs; however, the B‐conformation generally has more favorable van der Waals interactions than the A‐form. These sequence‐dependent conformational preference and environmental effects agree roughly with experimental observations, suggesting that the origin of the conformational polymorphism is attributable to the intrinsic conformational preference of base pairs.
Ca2+ binding cyclic octapeptides having an alternating sar and a hydrophobic amino acid in the sequence
Authors: Kimura Shunsaku , Ozeki Eiichi , Imanishi Yukio
Published: December 2004

Cyclic octapeptides having alternating Sar and hydrophobic amino acid sequences, such as cyclo[Lys(Z)‐Sar‐Leu‐Sar‐Leu‐Sar‐Leu‐Sar] (C8KL), cyclo[Glu(OMe)‐Sar‐Lys(Z)‐Sar‐Leu‐Sar‐Leu‐Sar] (C8KE), and cyclo[Lys(Suc)‐Sar‐Leu‐Sar‐Leu‐Sar‐Leu‐Sar][C8K(Suc)L, Suc represents succinic acid], were synthesized. These cyclic octapeptides formed a complex selectively with Ca2+. Upon complexation, trans peptide bonds of Sar residues were isomerized to cis peptide bonds. C8KL and C8KE showed very similar characteristics of Ca2+ binding, extraction of Ca2‐ from an aqueous solution to a chloroform solution, and Ca2+ transport through a liquid chloroform membrane. C8KL transported Ca2+ across the lipid bilayer membrane above the phase‐transition temperature, while C8KE and C8K(Suc)L did not. Therefore, the transport of Ca2+ through the lipid bilayer membrane is very sensitive to the hydrophobicity of the carrier molecule.
Ca2+ transport through lipid membrane by diastereomer cyclic octapeptides
Authors: Kimura Shunsaku , Ozeki Eiichi , Imanishi Yukio
Published: December 2004

Effects of the nature and orientation of a side chain in cyclic octapeptides on Ca2+ transport were examined by using cyclo[L‐Lys(Z)‐Sar‐LLeu‐Sar]2 (C8‐L), cyclo[L‐Lys(Z)‐Sar]4 (C8KS), and their diastereomer cyclic octapeptides, cyclo[L‐Lys(Z)‐Sar‐D‐Leu‐Sar]2 (C8‐D) and cyclo[L‐Lys(Z)‐Sar‐D‐Lys(Z)‐Sar]2 (C8Kk). All these cyclic octapeptides were found to take a single conformation in CDCl3, and the conformation was C2‐symmetric for C8‐L and C8‐D, and C4‐symmetric for C8KS and C8Kk. They formed a complex with Ca2+. Upon complexation, C8KS accompanied isomerization of peptide bonds, but C8‐D retained the arrangement of peptide bonds. The amount of Ca2+ extracted from an aqueous solution to a chloroform solution by all L cyclic octapeptide C8‐L or C8KS was about twice that of Na+, but 6 ∼ 8‐fold smaller than that by C8‐D or C8Kk including D units. These cyclic octapeptides were capable of transporting Ca2+ through a lipid membrane above the phase transition temperature, and the transport rate decreased in the order of C8Kk ∼ C8KS > C8‐D > C8‐L.
Two γ‐bends in the backbone conformation of [D‐ala2]‐leucine enkephalin in solution
Authors: Dhingra M. M. , Saran Anil
Published: December 2004

The solution conformation of [D‐Ala2]‐leucine enkephalin in its zwitterionic form in DMSO‐d6 has been monitored by one‐ and two‐dimensional proton magnetic resonance spectroscopy at 500 MHz. The resonances from the labile amide protons and the nonlabile protons have been assigned from the shift correlated spectroscopy. The chemical shift of the amide and C‐α protons are found to vary with temperature but in opposite directions, except the C‐α proton of the terminal tyrosine residue. This behaviour has been explained by the shifting of equilibrium between the zwitterionic and neutral forms of the [D‐Ala2]‐leucine enkephalin and probably conformational changes accompanying temperature variation. The low values of the temperature coefficients of leucine and glycine amide protons indicate that these protons are either intramolecularly hydrogen bonded or solvent shielded. The observation of sequential cross peaks in the nuclear Overhauser effect spectra obtained at various mixing times, τm (200–900 ms), indicate an extended backbone, which does not corroborate with the presence of a folded structure, i.e., β‐bend type structure. The estimate of interproton distances in conjunction with the low values of temperature coefficients of the leucine and glycine amide protons and vicinal coupling constants 3J have been rationalized by the predominance of two γ‐bends in the backbone conformation of [D‐Ala2]‐leucine enkephalin. The γ‐bend around the D‐Ala residue has Φ = 80° and ψ = 270°, while the one around Phe it has Φ = 285° and ψ = 90°.
A calorimetric approach to the study of the interactions of cytidine‐3′‐phosphate with bovine seminal ribonuclease
Authors: Ambrosino Rita , Barone Guido , Castronuovo Giuseppina , Cultrera Orazio , Di Donato Alberto , Elia Vittorio
Published: December 2004

A calorimetric study at 25°C is reported on the interaction between allosteric bovine seminal ribonuclease and cytidine‐3′‐phosphate. The results are compared with those obtained under identical experimental conditions for the interaction of pancreatic ribonuclease A and the same nucleotide. The analysis of the data provides evidence that the binding sites of seminal ribonuclease for cytidine‐3′‐phosphate are not equivalent, in agreement with previous equilibrium dialysis studies. A model with two sites with different affinities toward the nucleotide, the site with higher affinity resembling the binding site of pancreatic ribonuclease, is proposed. The values calculated for the thermodynamic parameters provide an insight of the forces involved in the interaction of the two enzymes with the nucleotide.
Long‐range structural effects in supercolied DNA: Statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion
Authors: Schaeffer Francis , Yeramian Edouard , Lilley David M. J.
Published: December 2004

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817–827] a set of sequences, called C‐type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C‐type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S‐type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations:

the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C‐type character;

C‐type induction by an A + T rich Drosophila sequence;

low‐temperature cruciform extrusion by an (AT)34 sequence;

the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat;

the effects of systematic deletion of the colL sequence; and

the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat.

These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.
The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling
Authors: Griess Gary A. , Moreno Elena T. , Easom Richard A. , Serwer Philip
Published: December 2004

By use of agarose gel electrophoresis, the sieving of spherical particles in agarose gels has been quantitated and modeled for spheres with a radius (R) between 13.3 and 149 mm. For quantitation, the electrophoretic mobility has been determined as a function of agarose percentage (A). Because a previously used model of sieving [D. Rodbard and A. Chrambach (1970) Proc. Natl. Acad. Sci. USA 65, 970–977] was found incompatible with some of these data, alternative models have been tested. By use of an underivatized agarose, two models, both based on the assumption of a single effective pore radius (PE) for each A, were found to yield PE values that were independent of R and that were in agreement with values of PE obtained independently (PE = 118 nm × A−0.74): sieving by altered hydrodynamics in a cylindrical tube of radius, PE, and sieving by steric exclusion from a circular hole of radius, PE. The same analysis applied to a 6.5% hydroxyethylated commercial agarose yileded a steeper PE vs A plot and also agreement of the above tow models with the data. The PE vs A plot was significantly altered by both further hydroxyethylation and factors that cause variation in the electro‐osmosis found in commercial agarose.
Orientational and rotational velocity correlation functions for water‐hydrating B‐ and Z‐DNA double helices
Authors: Swamy Kandadai N. , Clementi Enrico
Published: December 2004

The molecular dynamics simulations reported earlier for the structure and dynamics of water molecules hydrating B‐ and Z‐DNA double helices are analyzed for the orientational correlation functions and the proton rotational velocity autocorrelation functions. The spectra of the rotational velocity autocorrelation functions obtained from the simulation results are compared with the neutron inelastic scattering experiments on hydrated Na‐DNA samples. The results predict a small frequency component associated with water molecules bound to the double helices that disappears for waters away from the double helix.
Aggregation of amphotericin B in the presence of γ‐cyclodextin
Authors: Kajtár M. , Vikmon M. , Morlin E. , Szejtli J.
Published: December 2004

The macrolide antibiotic amphotericin B (AmB) forms an inclusion complex with γ‐cyclodextrin (γ‐CDx), resulting in a molecularly dispersed state of the drug. The state of aggregation of AmB in different solvents has been studied by absorption (uv‐vis) and CD spectroscopy. While in aqueous solutions AmB forms colloid‐like multimolecular aggregates, in the presence of γ‐CDx true solutions can be prepared, which show similar spectral properties as AmB dissolved in organic solvents. The AmB–γ‐CDx complex can be isolated as an amorphous, stable, water‐soluble powder, indicatin that γ‐CDx is a good carrier for the solubilization of this antibiotic. Using γ‐CDx as a carrier, the danger of precipitation of the drug during parenteral or intravenous administration can be largely reduced.
The CD of two‐chain coiled coils: Experiments on tropomyosin and tropomyosin segments in the tyrosin/disulfide spectral region
Authors: Holtzer Marilyn Emerson , Kumar Swatantar , Holtzer Alfred , Crimmins Dan L.
Published: December 2004

CD experiments are reported for several coiled‐coil species in the tyrosine/disulfide ( ∼ 250–350‐nm) region. Intact noncross‐linked tropomyosin ( ∼ 3°C) shows a negative nonsymmetric band maximal at 280 nm. This spectrum is the sum over six tyrosines/chain, and has conformational significance, since it disappears on denaturation. Experiments on an excised coiled‐coil segment, each of whose chains comprise residues 11–127 of the tropomyosin sequence and only one tyrosine (Y60), reveal that not all tyrosines are alike. The spectrum at 3°C shows a small negative maximum at ∼ 285 nm and a substantial, hitherto unknown, positive band at ∼ 270 nm, the latter masked in the parent protein by the negative contribution from the other tyrosines. A noncross‐linked coiled‐coil segment comprising residues 142–281, in which Y60 is absent, shows no such positive band. This peculiarity of Y60 is confirmed by absorbance spectra, with the extinction coefficient of Y60 larger in benign media than the average of the other tyrosines. Intact (3°C) C190 cross‐linked tropomyosin is known to yield, besides tyrosine contributions, a positive maximum at ∼ 300 nm. Subtracting the corresponding data for noncross‐linked tropomyosin shows that the disulfide spectrum itself actually has two equal, partly resolved bands at, respectively, 250 and 280 nm. The existence of a chiral disulfide argues for a relatively rigid, perhaps strained, local coiled coil. A C190 cross‐linked segment, comprising residues 142–281 shows a chiral disulfide spectrum like tropomyosin's, but another segment, comprising residues 168–284, shows none; thus removal of residues 142–167 causes loss of chirality at C190, over 20 residues away. These spectra thus contain important information on the subtle local differences in coiled‐coil structures.
A temperature‐dependent Z to B to single‐strand transition in d(CGCG)
Authors: Thomas Gerald A. , Peticolas Warner L.
Published: December 2004

A thermally induced left‐ to right‐handed helical interconversion for the self‐complementary tetradeoxynucleotide d(CGCG) has been observed in the presence of NaCl at concentrations from 1.5 to 2.25M. Analysis of the Raman spectrum of d(CGCG) in aqueous solution as a function of temperature indicates that at low temperature a left‐handed Z‐helical form is the predominant species. An increase in temperature results in a decrease in the population of the left‐handed form and an increase in the population of the right‐handed form. Further elevation in temperature results in extensive disruption of base stacking and a loss of secondary structure. This unstacking presumably represents dissociation to a single‐stranded structure. The data suggest that a temperature‐dependent Z to B to single‐strand transition occurs under the conditions employed. A direct Z to single‐strand conversion was not identified. In contrast to previous examples, the thermally induced left‐ to right‐handed helical conversion for d(CGCG) can occur in the absence of chemically modified nucleic acid bases, alcohol solutions, or divalent ions.
Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first‐order phase transition
Authors: Shakhnovich Evgeny I. , Finkelstein Alexey V.
Published: December 2004

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an “all‐or‐none” transition (independent of how compact is the denatured state of a protein and independent of the protein–solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free‐energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.
A CD determination of the α‐helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3
Authors: Clack Beatrice A. , Gray Donald M.
Published: December 2004

The CD spectra of four filamentous bacteriophages—fd, IKe, Pf1, and Pf3— were analyzed to determine the α‐helix contents of their major coat proteins. Measured spectra included the 192‐nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J. T. Yang, C.‐S. C. Wu, and H. M. Martinez (1986) Methods in Enzymology 130, 208–269] and (b) from poly(L‐lysine) [N. Greenfield and G. D. Fasman (1960) Biochemistry 8, 4108–4116]. Extended analyses were also performed with the addition of the spectrum of a model β‐turn to the Greenfield and Fasman reference set, with the spectrum of a short α‐helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L‐lysine) polypetide, plus a spectrum of a model β‐turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of α‐helix. The class I phages—fd and IKe—had large percentages of α‐helix of 98 ± 2 and 97 ± 5%, respectively, while the two class II phages—Pf1 and Pf3— had similar but smaller α‐helix contents of 83 ± 6 and 84 ± 2, respectively. While these α‐helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.
Nmr studies on the induced denaturation of lysozyme by guanidine hydrochloride in the presence of the inhibitor (NAG)3
Authors: Sheinblatt M.
Published: December 2004

The stabilization of the folded conformation of lysozyme, arising from the binding of the inhibitor (NAG)3 against induced denaturation, is demonstrated from the 1H‐nmr spectra of the enzyme. The nmr spectra reveal that the binding of the denaturant (GuHCl) to the enzyme is associated with changes in the conformation of the enzyme. The binding site of the inhibitor site C also serves as one of the binding sites of GuHCl. The observation that higher denaturant concentrations are required in the unfolding of Lys–(NAG)3 as compared to free Lys can be explained partly in terms of the existence of a competitive binding to the enzyme involving the (NAG)3 and GuHCl molecules.
Secondary structure of pig skin proteodermatan sulfate: A perspective from Raman spectroscopic studies in aqueous solution
Authors: Renugopalakrishnan V. , Zheng S. , Tu A. T. , Damle S. P.
Published: December 2004

Raman spectroscopic studies of pig skin proteodermatan sulfate in H2O are indicative of a well defined secondary structure consisting of α‐helical, β‐turn, and possibly “random” structures. The above conclusion is surprisingly close to the secondary structure of the “core” protein of pig skin proteodermatan sulfate proposed in the previous paper (V. Renugopalakrishnan et al., Biopolymers 28, 1923–1933, 1989) from FT‐IR and CD spectroscopic studies in aqueous solution.
Probe diffusion in cross‐linked actin gels
Authors: Newman Jay , Schick Kenneth L. , Zaner Ken S.
Published: December 2004

The diffusion of monodisperse polystyrene latex spheres (PLS) in column‐purified 0.65 mg/mL actin solutions, polymerized with 100 mM KCl in the absence and presence of a cross‐linker, actin binding protein (ABP), has been studied using dynamic light scattering. Measurements over a wide range of scattering angles from 90° to 8°, corresponding to inverse scattering vector probing distances of about 40–400 nm, respectively, give a measure of both the fraction of PLS mobile over the probing distance (from the normalized time autocorrelation function amplitude) and the average diffusion coefficient of the mobile PLS. Both 100‐ and 500‐nm diameter PLS are fully mobile in polymerized actin solutions over distances of less than 100 nm, as reported previously (Newman, J., Schick, K. S. & Gukelberger, G. Biophys. J. 53, 573a, and Newman, J., Mroczka, N. & Schick, K. L. Biopolymers, 28, 655–666). At increasing probing distances, or when ABP is added at molar ratios of 1 : 750 or 1 : 150, greater fractions of the PLS are immobilized, up to almost 99% at the conditions of a 400‐nm probing distance with 500‐nm probes and at a ratio of 1 : 150 added ABP to actin. The degree of immobilization correlates well with the amount of added ABP, the size of the PLS, and the probing distance. At increasing probe distances, as the degree of immobilization increases, the remaining mobile fraction of PLS has an increasing average diffusion coefficient. These results suggest a range of pore size in the actin gels with a mean size of a few hundred nanometers.
The effects of pH on hyaluronate as observed by light scattering
Authors: Reed Christopher E. , Li Xiao , Reed Wayne F.
Published: December 2004

Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low‐power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well‐behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation.
Fourier transform infrared spectroscopy for the characterization of a model peptide–DNA interaction
Authors: Dev S. B. , Walters Lee
Published: December 2004

To better understand the structural basis of protein–DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29‐residue synthetic peptide binds nonspecifically to heterogenous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100–200 μM produces 50% saturation of heterogenous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second‐derivative spectra resolve the amide I band of native peptide into components located at 1627 (β‐strand), 1658 (α‐helix), and 1681 (turn or β‐strand) cm−1, with a distinct shoulder at 1647 cm−1 (disordered structure). Assignment of the 1681 cm−1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and β‐strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm−1 (turn) is no longer seen; a new band appears at 1675 cm−1; the 1627 cm−1 band (β‐strand) is considerably reduced in intensity; the position of the α‐helical (1658 cm−1) component remains unchanged; the shoulder at 1647 cm−1 (disorder) disappears. The new vibration at 1675 cm−1 is characteristic of β‐strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm−1 (bound); the relative intensities of νAS and the PO symmetric stretch (vs) are altered upon peptide binding. The data is consistent with the following conclusions: (a)DNA binding changes the secondary structure of the peptide, (b) disordered region(s) are only observed in free peptide, i.e., DNA binding stabilizes and increases order in the peptide secondary structure, (c) turn(s) change into β‐strand and/or α‐helical conformations(s) when peptide binds to DNA, (d) a β‐strand conformation that is characterized bya 1627 cm−1 vibration is present in free and bound peptide, (e) there is a particular β‐strand vibration/conformation that is only present in the bound peptide, (f) an α‐helical region exists in both free and bound peptide, (g) DNA remains in a B‐family conformation upon peptide binding, and (h) DNA phosphates participate in peptide binding and/or subtle changes occur in the DNA conformation upon complex formation.
Range of the influence of the carbohydrate moiety on the conformation of the poly(amino acid) backbone in glycosylated mucins
Authors: Tanpipat Noppawan , Mattice Wayne L.
Published: December 2004

The influence of glycosylation on the conformational properties of porcine submaxillary gland mucin has been investigated using rotational isomeric state theory. The specific objective was to determine the conditions under which the polypeptide has the relatively large mean square unperturbed radius of gyration 〈s2〉0, demanded by the measurements of Shogren et al., while retaining the overall architecture of a random coil. The mean square dimensions were monitored as the dimensionless characteristic ratio defined as C = 〈s2〉0/npl, and the overall architecture was monitored by another dimensionless ratio 〈r2〉0/〈s2〉0, where 〈r2〉0 denotes the mean square unperturbed end‐to‐end distance. The computed values of C cannot reproduce the measured values if the conformational influence of glycosylation is restricted to each Ser or Thr, or if this influence extends only as far as their nearest neighbors. Values of C compatible with experiment can be obtained if the influence extends to next nearest neighbors. The behavior of the computed values of 〈r2〉0/〈s2〉0 permits an assignment of 7 ± 1 as the likely upper limit to the number of consecutive amino acid residues that experience alterations in ϕ and ψ if the sequence contains a glycosylated Ser or Thr.
Electrostatic forces at helix–coil boundaries in DNA
Authors: Blake R. D. , Delcourt Scott G.
Published: December 2004

The Tm of internal loop‐forming (dA · dT)N domains in pBR322 DNA has been measured over a tenfold range of [Na+]. The slopes SN = dTm/d log[Na+] are linear and decrease in magnitude with decreasing loop size N, signaling a reduction in Na+ released during the transition of these domains to the coil state. Values of SN decrese linearly with increasing N−1 in accordance with the expectation of a simple model for the occurrence of a gradient of long‐range electrostatic forces at helix–coil boundaries, and extrapolate almost precisely to the value of S∞ observed for (dA · dT)∞. These results indicate (1) less counterion is released per phosphate residue from the finite loop than from the infinitesized loop, and (2) the difference in binding is constant for each boundary formed and independent of the size of the loop within the range examined: ∼ 350 base pair (bp) > N > 71 bp. The slope of the dependence of SN on N−1 indicates the region of higher charge density at the boundary extends at least 18 Å into the coil and probably 40–50 Å before dropping to a value characteristic of the unperturbed coil. The free energy for excess counterion binding at boundaries can be expressed by

When the loop entropy function in a statistical mechanical algorithm for the dissociation of DNA is weighted by this quantity, calculated Tm are seen to vary by only ±0.09°C from observed.
Assignment of the 13C‐nmr spectra of virgin and reactive‐site modified turkey ovomucoid third domain
Authors: Robertson A. D. , Rhyu G. I. , Westler W. M. , Markley J. L.
Published: December 2004

The virgin (reactive‐site Leu18‐Glu19 peptide bond intact) and modified (reactive‐site Leu18‐Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton‐detected 1H{13C} two‐dimensional single‐bond correlation (1H{13C}SBC) spectroscopy. Previous 1H‐nmr assignments of these proteins [A. D. Robertson, W. M. Westler, and J. L. Markley (1988) Biochemistry, 27, 2519–2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529–2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13Cα‐1H units and numerous side‐chain 13C‐1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side‐chain prochiral methylene protons attached to the Cγ of Glu19 and Cδ of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shift in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18‐Glu19 peptide bond. Comparison of 13Cα chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839–5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557–7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133–2141] suggests that 13Cα chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.
The elastic modulus of fibrin clots and fibrinogen gels: The effect of fibronectin and dithiothreitol
Authors: Procyk R. , King R. G.
Published: December 2004

The elastic modulus (G′) of factor XIIIa induced fibrinogen gels was found to be substantially lower than the G′ of fibrin gels that were formed by clotting fibrinogen with thrombin. The addition of fibronectin and/or the reducing reagent dithiothreitol (DTT) to the factor XIIIa coagulation mixture led to the formation of a weaker gel structure, while the rigidity of thrombin induced clots was not appreciably affected by the inclusion of the DTT but increased somewhat in the presence of fibronectin. The reasons for the differing clot rigidities are discussed in terms of biochemical mechanisms.
Computational analysis of the effects of site‐specific phosphate alkylation in the DNA oligomer {d‐[GGAATTCC]}2
Authors: Broido Michelle S. , Mezei Mihaly
Published: December 2004

Alkylation of the sugar–phosphate backbone of DNA can result upon exposure to several potent carcinogens, inducing DNA misfunction. In order to assess the structural and energetic changes in DNA helices induced by such alkylation, we have performed AMBER‐based analyses on phosphotriester containing analogues of {d‐[GGAATTCC]}2. Fourteen analogues of the nonalkylated oligomer were examined, each bearing a single alkylation of known stereochemistry. Results indicate that although there is minimal effect on the aromatic bases, the presence of a phosphotriester disturbs the sugar–phosphate backbone in complex ways. For most analogues, total minimum energies are lower for the Sp‐alkylations than for the Rp‐alkylations which point directly into the major groove of the helix; however, different energetic contributions follow different, or no, trends in dependence on alkylation site and/or stereochemistry. Where data is available, experimental nmr results agree with the calculations reported here.
Free energy calculation on base specificity of drug – DNA interactions: Application to daunomycin and acridine intercalation into DNA
Authors: Cieplak Piotr , Rao Shashidhar N. , Grootenhuis Peter D. J. , Kollman Peter A.
Published: December 2004

We present the results of free energy perturbation/molecular dynamics studies on B‐DNA · daunomycin and B‐DNA · 9‐aminoacridine complexes as well as on B‐DNA itself in order to calculate the free energy differences between complexes having different base pair sequences. The results generally reproduce the trends observed experimentally, i.e., preferences of acridine and daunomycin to bind to a specific base sequence in the DNA. This is encouraging, given the simplicity of the molecular mechanical/dynamical model in which solvent is not explicitly included.
Energetics of intrachain salt‐linkage formation in collagen
Authors: Katz E. P. , David C. W.
Published: December 2004

The energy of formation of salt linkages between Arg or Lys with Asp or Glu in a polypeptide chain having the collagen fold have been estimated using the fully empirical energy minimization scheme AMBER. The polypeptide was considered both in an isolated and a hydrated triple helical state. The collagen fold associated with a one‐bonded triple helical conformation allows intrachain salt linkages having stabilization energies of 60–100 kcal when the reacting residues are separated by no more than two intervening residues. The amino end of one side chain always approaches the carboxyl end of the other side chain, and simultaneously approaches the carbonyl oxygen of the intervening backbone residue. The salt linkage conformation and the backbone conformation of the isolated collagen fold in vacuo are maintained when the molecules are in a hydrated triple helix. These results are compatible with a fold‐forming role for salt linkages, especially in proline poor regions, during collagen polypeptide synthesis, and with the persistence of intrachain salt linkages throughout molecular and fibril assembly.