bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of the folding of short polypeptide segments by uniform conformational sampling
Authors: Bruccoleri Robert E. , Karplus Martin
Published: December 2004

A procedure, CONGEN, for uniformly sampling the conformational spaceof short polypeptide segments in proteins has been implemented. Because thetime required for this sampling grows exponentially with the number of residues, parameters are introduced to limit the conformational space that has to be explored. This is done by the use of the empirical energy function ofCHARMM [B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan and M. Karplus (1983) J. Comput. Chem. 4, 187‐217] and truncating the search when conformations of grossly unfavorable energy are sampled. Tests are made to determine control parameters that optimize the search without excluding important configurations. When applied to known protein structures, the resulting procedure is generally capable of generating conformations where the lowest energy conformation matches the known structure within a rms deviation of 1 Å.
A theoretical exploration of conformational aspects of ethidium bromide intercalation into a d(CpG)2 minihelix
Authors: Chen Kai‐Xian , Gresh Nohad , Pullman Bernard
Published: December 2004

In order to gain insight into the structural and conformational factors involved in the binding of ethidium bromide to oligonucleotides, theoretical computations are performed on the ethidium bromide–d(CpG)2 complexes, in which the ligand–nucleotide interaction energy, the conformational energy of the ligand, and that of the nucleotide are simultaneously optimized. The intermolecular and conformational energy computations are performed with the SIBFA procedures (sum of interactions between fragments computed ab initio), which use empirical formulas based on ab initio SCF (Self Consistent Field) computations. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structure) elaborated by Miller and co‐workers. The pyrimidine sugar is held in the C3′‐endo conformation and the purine sugar in the C2′‐endo conformation in conformity with both x‐ray and nmr results for dinucleoside duplexes. Several candidate intercalation sites, selected on the basis of energy criteria, could be generated by the procedure. They were found to recur in a persistent fashion over the whole range of unwinding angles investigated, which span from −20° to −30°. Among these, a conformation closely similar to the one determined crystallographically by Sobell et al. was found to be the intrinsically most favorable and to react with the strongest interaction energy with ethidium. For this conformation, the most favorable value of the unwinding angle is −26°, close to the value in the crystal structure. Several pairwise correlations of structural parameters could be evidenced. The effect of the 2′OH groups (as in RNA duplexes as opposed to DNA duplexes) was also investigated and shown to further enhance, albeit slightly, the intrinsic stability of the Sobell‐like Δα = −26° conformation.
On the stretching of DNA in the reptation theories of gel electrophoresis
Authors: Slater Gary W. , Rousseau Jean , Noolandi Jaan
Published: December 2004

The reptation dynamics of DNA chains in gel electrophoresis is discussed. The differences between the Lumpkin, Déjardin, and Zimm (LDZ), and the Slater and Noolandi (SN), biased reptation models are pointed out and studied. We show that new assumptions are necessary in order to study large‐field electrophoresis, and we present preliminary results of a computer simulation of DNA gel electrophoresis.
Protofibrin clots induced by calcium and zinc
Authors: Marx Gerard
Published: December 2004

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin‐activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration‐dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)‐ or Zn(II)‐induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation‐sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.
Monte carlo studies on equilibrium globular protein folding. I. Homopolymeric lattice models of β‐barrel proteins
Authors: Kolinski Andrzej , Skolnick Jeffrey , Yaris Robert
Published: December 2004

Dynamic Monte Carlo studies have been performed on various diamond lattice models of β‐proteins. Unlike previous work, no bias toward the native state is introduced; instead, the protein is allowed to freely hunt through all of phase space to find the equilibrium conformation. Thus, these systems may aid in the elucidation of the rules governing protein folding from a given primary sequence; in particular, the interplay of short‐ vs long‐range interaction can be explored. Three distinct models (AC) were examined. In model A, in addition to the preference for trans (t) over gauche states (g+ and g−) (thereby perhaps favoring β‐sheet formation), attractive interactions are allowed between all nonbonded, nearest neighbor pairs of segments. If the molecules possess a relatively large fraction of t states in the denatured form, on cooling spontaneous collapse to a well‐defined β‐barrel is observed. Unfortunately, in model A the denatured state exhibits too much secondary structure to correctly model the globular protein collapse transition. Thus in models B and C, the local stiffness is reduced. In model B, in the absence of long‐range interactions, t and g states are equally weighted, and cooperativity is introduced by favoring formation of adjacent pairs of nonbonded (but not necessarily parallel) t states. While the denatured state of these systems behaves like a random coil, their native globular structure is poorly defined. Model C retains the cooperativity of model B but allows for a slight preference of t over g states in the short‐range interactions. Here, the denatured state is indistinguishable from a random coil, and the globular state is a well‐defined β‐barrel. Over a range of chain lengths, the collapse is well represented by an all‐or‐none model. Hence, model C possesses the essential qualitative features observed in real globular proteins. These studies strongly suggest that the uniqueness of the globular conformation requires some residual secondary structure to be present in the denatured state.
Time‐Resolved fluorescence polarization anisotropy of short restriction fragments: The friction factor for rotation of DNA about its symmetry axis
Authors: Wu Pengguang , Fujimoto Bryant S. , Michael Schurr J.
Published: December 2004

The time‐resolved fluorescence polarization anisotropy (FPA) of ethidium dye intercalated in 43 and 69 base pair (bp) restriction fragments is measured, and the friction factor per bp for rotation of DNA about its symmetry axis is determined. The same value of the hydrodynamic radius, a = 12.0 ± 0.6 Å, is obtained for both the 43‐ and 69‐bp fragments, but only when (1) the twisting correlation functions appropriate for such short filaments are used: (2) the correct amplitude is employed for the uniform tumbling mode decays: and (3) the data analysis is restricted to times after the internal bending modes have died away leaving just reduced amplitudes of the exponentially decaying uniform tumbling modes. The present value of the hydrodynamic radius is significantly larger than that implied by the cross‐sectional area perpendicular to the symmetry axis. This strongly suggests that a significant fraction of water in the major and minor groves is rotating more or less rigidly with the DNA on this time scale. The correct expression for the amplitude Dn(∞) of the uniform mode decay of the tumbling correlation function, including the average over all sites to which the dye could bind, is derived in the appendix. The present theory for Dn(∞) is compared with that of Barkley and Zimm [(1979) J. Chem. Phys. 70, 2991–3007], and with recent Brownian simulations of discrete wormlike chains by Allison and co‐workers [S. A. Allison and J. A. McCammon (1984) Biopolymers 23, 363–375; S. A. Allison (1986) Macromolecules 19, 118–124].
The thermodynamic parameters of the interaction of concanavalin A with glycosyl‐free liposomes: A microcalorimetric study
Authors: Lüscher‐Mattli Madeleine
Published: December 2004

The nonspecific interaction of the mitogenic lectin Concanavalin A (Con A) with glycosyl‐free liposomes of various composition has been investigated by microcalorimetric titration measurements. The results obtained show the following features of main interest: (1) the affinity constants (Ka) of the interaction of Con A with liposomal bilayers are in the order of magnitude 105–106M−1. The reaction enthalpies (ΔH) are positive, and small (approximately 0.1 KJ mol−1 lipid), compared to the free energy terms (−ΔG = 30–40 KJ mol−1 lipid). All lectin–lipid interactions are strongly entropy‐controlled (ΔH/TΔS < 1.0). These thermodynamic features are characteristic for hydrophobic interaction processes. (2) The liposomal head‐group charge does not significantly affect the lipid‐affinity of Con A. Electrostatic forces thus appear to play a minor role in lectin–lipid interactions. (3) The lipid affinity of Con A is sensitive to the fluidity of the liposomal bilayers, increasing with increasing fluidity. Below the gel to liquid‐crystal phase transition temperature, the lectin binding to liposomal bilayers is inhibited. (4) The binding isotherms, corresponding to the interaction of Con A with liposomes, composed of tightly packed, saturated phospholipids, exhibit pronounced positive cooperativity. This phenomenon is absent in the binding curves, corresponding to the interaction of Con A with more fluid liposomal bilayers. (5) The Con A specific inhibitor α‐D‐methylmannopyranoside (50 mM) drastically increases the molar reaction enthalpy. The Ka term is significantly reduced in presence of the inhibitor sugar. Urea induces analogous changes in the thermodynamic parameters of the lectin–lipid interaction. The effects of α‐D‐methylmannopyranoside are thus not Con A specific, but are attributable to solvent effects. (6) It was shown that the binding of one Con A molecule affects a large number (approximately 1000) of phospholipid molecules in the liposomal bilayer. (7) The affinity constants (Ka) of the interaction of Con A with glycosyl‐free lipids are smaller by a factor of approximately 10, compared to the Ka terms, reported for Con A binding to biological membranes. The presence of glycosidic receptor groups thus controls the specificity of lectin–membrane interactions, whereas the nonspecific lectin–lipid interactions appear to represent the main driving force for the strong attachment of the lectin to membrane surfaces.
β‐Turns in bridged proline‐containing cyclic peptide models
Authors: Hollósi M. , Kövér K. E. , Holly S. , Radics L. , Fasman G. D.
Published: December 2004

The synthesis, CD, ir spectroscopic, and conformational studies of a series of bridged cyclic peptides of the general formula, cyclo[NH‐(CH2)n‐CO‐Gly‐Pro‐Y‐Gly] (2a–d, Y = Gly or Ser(OBut), n = 4 or 2) is reported. As indicated by difference nuclear Overhauser enhancement and Fourier transform ir experiments, the tetrapeptide sequence of cyclo[NH‐(CH2)4‐CO‐Gly‐Pro‐Gly‐Gly] (2a) and cyclo[NH‐(CH2)2‐CO‐Gly‐Pro‐Gly‐Gly] (2b) adopts a 1 ← 4 hydrogenbonded type II β‐turn conformation in solution, while cyclo[NH‐(CH2)4‐CO‐Gly‐Pro‐Ser(OBut) ‐Gly] (2c) features a type I β‐turn, fixed by 1 ← 4 and Oγ … NH intramolecular H bonds. In aqueous solution 2a and 2c show class B and class C CD spectra, respectively. This is the first case reported of a typical class C CD pattern in aqueous solution for a conformationally mobile system having a type I β‐turn. Based on the comparison of the band intensities of the bridged models with those of linear and cyclic model systems reported earlier, a set of subspectra with reduced band intensities is suggested for use in the CD analysis of the conformation of polypeptides in solution.
Proline‐induced constraints in α‐helices
Authors: Piela Lucjan , Némethy George , Scheraga Harold A.
Published: December 2004

The disrupting effect of a prolyl residue on an α‐helix has been analyzed by means of conformational energy computations. In the preferred, nearly α‐helical conformations of Ac‐Ala4‐Pro‐NHMe and of Ac‐Ala7‐Pro‐Ala7‐NHMe, only the residue preceding Pro is not α‐helical, while all other residues can occur in the α‐helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α‐helix. Other low‐energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near‐α‐helical A‐conformational state (with up to 26° changes of their dihedral angles ϕ and ψ, and a 48° change in one ω from those of the ideal α‐helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α‐helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α‐helices, 61 have a Pro at or next to their N‐terminus, 21 have a Pro next to their C‐terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α‐helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.
Dynamic light‐scattering study of semiflexible polymers: Collagen
Authors: Kubota Kenji , Tominaga Yasunori , Fujime Satoru
Published: December 2004

Dynamic light‐scattering measurements were carried out for collagen in acetate buffer (pH 4.8) extracted from lathyritic ratskin. The correlation functions were analyzed in terms of the semiflexibility of collagen molecules. The experimental Γ/K2 vs K2 relationship was compared with the theoretical one based on formulation including anisotropy in translational diffusion, chain flexibility, and the hydrodynamic interaction; Γ is the average decay rate and K is the magnitude of the momentum transfer vector. By using the model parameters evaluated from the Γ/K2 vs K2 relationship, a good agreement was obtained between profiles of theoretical and experimental correlation functions over the entire delay times. Detailed examinations of the dynamic light‐scattering spectrum permitted us to conclude that a set of the contour length L of 300 nm and the Kuhn length γ−1 of 340 nm are much more probable than other sets of L and γ−1 that equally explain static quantities such as the radius of gyration. The results show that collagen molecules are well characterized by a wormlike chain model.
Simulation of the diffusion‐controlled reaction between superoxide and superoxide dismutase. II. Detailed models
Authors: Allison S. A. , Bacquet R. J. , McCammon J. A.
Published: December 2004

A two‐stage Brownian dynamics simulation method is used to study the diffusion‐influenced bimolecular reaction between superoxide and superoxide dismutase (SOD). The crystal structure of the dimeric enzyme is used in constructing detailed topographical and electrostatic models. Several electrostatic models are considered. In the most realistic, the excluded volume of the protein, which is impermeable to penetration by mobile ions, is assigned a dielectric constant of 2 and the surrounding “solvent” is assigned a value of 78. A finite difference method is used to solve the linearized Poisson–Boltzmann equation. For native SOD, the simulations reproduce the pronounced salt dependence of the rate constant observed experimentally. This salt dependence is attributed to electrostatic interactions between enzyme and substrate that are inherently attractive and amplified by the low dielectric constant of the protein interior. The simulation method is also applied to a modified enzyme, acylated SOD.
The role of hydroxyproline in collagen folding: Conformational energy calculations on oligopeptides containing proline and hydroxyproline
Authors: Bansal M. , Ananthanarayanan V. S.
Published: December 2004

We have observed that the rate of folding of the enzymatically hydroxylated form of poly(Gly‐Pro‐Pro) into the triple‐helical conformation is considerably higher than that of the unhydroxylated polypeptide [R. K. Chopra and V. S. Ananthanarayanan (1982) Proc. Natl. Acad. Sci. USA 79, 7180–7184]. In this study, we examine a plausible kinetic pathway for triple‐helix formation by selecting peptide models for the unhydroxylated collagen molecule, and computing their conformational energies before and after proline hydroxylation. Starting with the available data on the preferred conformations of proline‐ and hydroxyproline‐containing peptide sequences, energy minimization was carried out on the following pairs of peptides: Gly‐Ala‐Pro‐Gly‐Ala and Gly‐Ala‐Hyp‐Gly‐Ala; Gly‐Pro‐Pro‐Gly‐Ala and Gly‐Pro‐Hyp‐Gly‐Ala; Gly‐Ala‐Pro‐Gly‐Ala‐Pro and Gly‐Ala‐Hyp‐Gly‐Ala‐Hyp. It was found that, with each pair of peptides, the energetically most favorable conformation (I) has an extended structure at the Gly‐Ala or Gly‐Pro segment and a β‐bend at the Pro‐Gly or Hyp‐Gly segment. In the Hyp‐containing peptides, this conformation is further stabilized by a (Hypi + 2)OH…OC(Glyi) hydrogen bond. Conformation I is lower in energy by about 6–13 kcal/mol of the peptide than the fully extended conformations that resemble the single collagen polypeptide chain and contain no intramolecular hydrogen bond. In contrast to the proline counterpart, the hydroxyproline‐containing peptides are found capable of adopting a partially extended conformation that does not contain the β‐bend but retains the (Hyp)OH…OC(Gly) hydrogen bond. The energy of this conformation is intermediate between conformation I and the fully extended conformation. The continuation of the β‐bend along the chain is restricted by stereochemical constraints that are more severe in the latter two pairs of peptides than in the first pair. Such a restriction may be considered to trigger the “unbending” of the minimum energy conformation leading to its straightening into the fully extended conformation; the latter, in turn, would lead to triple‐helix formation through favorable interchain interactions. We propose that the partially extended conformation in the Hyp‐containing peptides could serve as a kinetic intermediate on the way to forming the fully extended conformation. Because of the (Hypi + 2)OH…OC(Glyi) hydrogen bond, this conformation would also serve to lock the trans geometry at the Gly‐Ala(Pro) and Ala(Pro)‐Hyp peptide bonds, thereby enhancing the rate of their helix formation. A scheme for collagen folding in proposed on the basis of these results.
Orientation of DNA during gel electrophoresis studied with linear dichroism spectroscopy
Authors: Jonsson Mats , Åkerman Björn , Nordén Bengt
Published: December 2004

A method for in situ study of orientation of DNA during gel electrophoresis has been developed. Linear dichroism spectra measured by this phase‐modulation technique can sensitively and selectively detect orientation of DNA during electrophoretic migration in gel. [Measurement of “electrophoretic orientation” was first reported in 1985 by B. Åkerman, M. Jonsson, and B. Nordén (1985) (J. Chem. Soc. Chem. Commun. 422–423)]. Restriction fragments of duplex DNA of lengths in the ranges of 300–2319 base pairs (bp) and 4361–23130 bp have been studied in 5% polyacrylamide and 1% agarose gels, respectively. The fragments become preferentially oriented with the DNA helix axis parallel to the migration direction. In agarose the orientation is found to increase sigmoidally, and in polyacrylamide, linearly, with the electric field strength, within the field ranges accessible to measurement (0–40 and 5–40 V/cm, respectively). In both types of gels a considerable increase in orientation with length of DNA was observed. Compared to dipole orientation in electric fields, the electrophoretic orientation is high: orientation factor S = 0.027 in agarose for 23130 bp at 10 V/cm and S = 0.004 in polyacrylamide for 2319 bp at 10 V/cm. In addition to orientation of DNA, the electrophoresis also leads to orientation effects in the gel structure owing to Joule heating. In agarose there is also an effect that is associated with the migrating DNA zones and that produces different orientations of the gel at the front and rear parts of a zone. Evidence is presented that this effect is due to a DNA‐induced electroosmotic flow causing a contraction of the gel in the front of the zone and an expansion in the rear. The experimental results on DNA orientation are compared with the reptation theories for gel electrophoresis. The theory of Lumpkin et al. [O. J. Lumpkin, P. Dejardin, and B. H. Zimm (1985) Biopolymers 24, 1573–1593] predicts no orientation length dependence, but it does predict a shape of the field dependence that resembles the shape observed in agarose. The theory of Slater and Noolandi [G. W. Slater and J. Noolandi (1986) Biopolymers 25, 431–454] predicts an orientational length dependence that is an order of magnitude less than the experimental one, and a field dependence that agrees neither with the sigmoidal shape observed in agarose nor with the linear dependence in polyacrylamide.
A type II β‐turn in a flexible peptide: Proton assignment and conformational analysis of the α‐factor from saccharomyces cerevisiae in solution
Authors: Jelicks Linda A. , Naider Fred R. , Shenbagamurthi Ponniah , Becker Jeffrey M. , Broido Michelle S.
Published: December 2004

Two‐dimensional 1H‐nmr spectra of the α‐mating factor [in dimethyl sulfoxide‐d6 (DMSO) and in water] and several dodecapeptide analogues (in DMSO) were obtained. Homonuclear correlated spectroscopy resulted in the complete and unequivocal assignment of all backbone and side‐chain resonances of the peptides. The solution conformation of the pheromones was probed using two‐dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauer effect spectroscopy (ROESY). The 2D NOE results, and results of complementary one‐dimensional experiments, suggest that a type II β‐turn is assumed by the central portion of active pheromones in both DMSO and water. Inactive analogues of the α‐factor do not exhibit this structural feature. Except for this one β‐turn, the nmr parameters for α‐factor are indicative of a conformationally flexible molecule in both solvents. This conclusion is in contrast to that of other researchers who have proposed a highly structured conformation of α‐factor in aqueous solution.
Prediction of protein helix content from an autocorrelation analysis of sequence hydrophobicities
Authors: Horne David S.
Published: December 2004

It is demonstrated that protein α‐helix content can be predicted from an autocorrelation analysis of the protein hydrophobicity sequence. The Fourier transform of the autocorrelation function yields the spectral densities or weights of the various frequencies contributing to the autocorrelation function. Using sequence and secondary structure data from more than 160 proteins and domains, a linear relationship was found between spectral density at periodicity 3.7 and protein α‐helix content (r = 0.83). This relation permits prediction of the helix content (x) of proteins of known sequence to within ± 15%, i.e., as (x ± 15)%. Predictions based on the autocorrelation procedure are compared with values obtained by other methods.
Conformations and mitochondrial uncoupling activity of synthetic emerimicin fragments
Authors: Raj P. Antony , Das Manoj K. , Balaram P.
Published: December 2004

Several amino terminal fragments of the emerimicins (Ac‐Phe1‐Aib2‐Aib3‐Aib4‐Val5‐Gly6‐Leu7‐Aib8‐Aib9‐Hyp10‐Gln11‐D‐Iva12‐Hyp13‐Ala/Aib14‐Phol15) ranging in length from five to ten residues have been synthesized. Nuclear magnetic resonance studies have been carried out on the 1‐5, 6‐10, 1‐6, 1‐7, 1‐8, 1‐9, and 1‐10 fragments. The number of solvent‐shielded NH groups in CDCl3 solutions for 1‐5, 1‐6, 1‐7, 1‐8, 1‐9, and 1‐10 indicate that 310‐helical structures are favored in this solvent. In (CD3)2SO, an additional NH group, assigned to Aib(3) NH is solvent exposed in the fragments longer than six residues, suggesting partial unfolding of the N‐terminal β‐turn or transition to an α‐helical conformation. The data for fragment 6‐10 are consistent with a conformation having a single Leu‐Aib β‐turn. Infrared studies suggest an increase in the number of intramolecular hydrogen bonds with increasing peptide chain length. Appreciable mitochondrial uncoupling activity is observed for peptides with a chain length of at least seven residues. The order of efficiencies of the fragments is 1‐7 < 1‐8 ∼ 1‐10 < 1‐9, with the decapeptide exhibiting anomalously low uncoupling activity.
Vibrational CD of polypeptides. XII. Reevaluation of the fourier transform vibrational CD of poly(γ‐benzyl‐L‐glutamate)
Authors: Malon Petr , Kobrinskaya Rina , Keiderling Timothy A.
Published: December 2004

Using a newly constructed Fourier transform ir (FTIR) based vibrational CD (VCD) instrument, we have found that elimination of the ellipsoidal collection mirror before the detector and its replacement by a lens leads to a significant improvement in the absorption artifact problem often seen previously in FTIR‐VCD. Reduction in artifact level brings the FTIR‐VCD band shape of the amide 1 band in poly(γ‐benzyl‐L‐glutamate) into better agreement with that found with dispersive instruments. These results indicate that the difference between the spectra obtained with the two methods is not primarily a factor of resolution but is more dependent on artifact level. Thus the previously reported deconvolution of the amide I VCD results and their subsequent interpretation in terms of the helical vs. exciton mechanisms overemphasize weak features in the spectrum. Results based on deconvolution of the absorption spectrum remain valid. Further new data on the amide II and several lower energy transitions that encompass the amide III region show broad single‐signed features—but of the opposite sense—in the two regions, implying that their VCD arises through mutual coupling.
On the interaction of chromomycin A3 with calf thymus DNA in the presence of metal cations at different pH values
Authors: Weinberger Sarah , Shafer Richard , Berman Elisha
Published: December 2004

The interactions of the antitumor antibiotics, chromomycin A3, with a variety of metal cations in the pH range of 3.0–8.5 were systematically studied by CD, absorption, and 1H‐nmr spectroscopies. Results were compared with those obtained in the presence of increasing amounts of calf thymus DNA. The negatively charged chromomycin A3, pKa 6.3, forms aggregates that become ordered and smaller in size, in the presence of variety of metal cations. Spectrophotometric titrations have shown that binding of the neutral drug to DNA at pH 4.5 does not require divalent cations, although the strength of the binding is greatly enhanced in their presence. At higher pH values (> 7.0) and low DNA/drug ratio ( > 20), the metal cations are necessary to induce the binding between chromomycin A3 and DNA. At higher DNA/drug ratios (> 100: 1), an appreciable proportion of the drug is bound even in the absence of divalent cations. Its binding affinity to the DNA is enhanced in the presence of these cations and at low pH values. Therefore, we conclude that chromomycin A3 binds in two related modes, in the presence and in the absence of divalent cations. The spectral data accumulated indicate the metal cation is involved in the binding of the drug to the DNA by forming a drug–metal–DNA ternary complex.
The association behavior of β‐lactamases in polyethylene glycol solution
Authors: Murthy N. Sanjeeva , Braswell Emory H. , Knox James R.
Published: December 2004

The β‐lactamases (EC 3.5.2.6) from Bacillus licheniformis 749/C, Enterobacter cloacae P99, and TEM plasmid RP4 are studied in 10–14% (w/v) polyethyleneglycol (PEG) 8000 solutions at pH 6.5 by x‐ray scattering and in 18% PEG by equilibrium sedimentation. Although all three enzymes crystallize with twofold crystal symmetry from PEG 8000, it is not possible in this study to prove that dimerization occurs; however, both techniques give evidence for association above 1% (w/v) protein concentration. For the B. lichen., P99, and TEM enzymes, a dimerization of at most 0, 5, and 10% (v/v), respectively, account for the variation of radii of gyration Rg with concentration, after accounting for the effects of nonideality. Apparent Rg were 3–5% smaller in PEG solution than in PEG‐free solution. Enhanced ordering of the molecules in PEG solution or the presence of a PEG‐depleted hydration shell around the enzymes can account for the observation of reduced Rg values. Accordingly, values of the partial specific volume (defined at constant chemical potential of PEG) indicate considerable PEG exclusion and are consistent with the ability of high Mr PEGs to induce crystallization of these enzymes.
Vasopressin conformational fluctuations: A molecular dynamics study
Authors: Somoza J. R. , Brady J. W.
Published: December 2004

Molecular dynamics simulations have been used to study the conformational fluctuations of the oligopeptide hormone vasopressin. Starting coordinates for these simulations were built upon the crystal structure of pressinoic acid, the cyclic ring moiety of vasopressin, recently determined by x‐ray diffraction. Coordinates for the additional tripeptide “tail” of vasopressin were selected by arbitrary positioning of this segment using interactive computer graphics. Two such starting configurations were minimized to relax strains, and long dynamics simulations (20 and 40 ps) in vacuo were then conducted following extensive heating and equilibration sequences (36 ps). In these studies, vasopressin was found to undergo few substantial conformational changes at 300 K on the time scale simulated, in contrast to the results of a shorter previous simulation, but comparable structural transitions were observed during the equilibration periods. The pressinoic acid structure was found to be a reasonably stable possible conformation for vasopressin in vacuum on this time scale.
The origin of the A to B transition in DNA fibers and films
Authors: Lindsay S. M. , Lee S. A. , Powell J. W. , Weidlich T. , Demarco C. , Lewen G. D. , Tao N. J. , Rupprecht A.
Published: December 2004

We have studied the hydration of Na‐DNA and Li‐DNA fibers and films, measuring water contents, x‐ray fiber diffraction patterns, low‐frequency Raman spectra (below 100 cm−1), high‐frequency Raman spectra (600–1000 cm−1), and swelling, as a function of relative humidity. Most samples gain weight equilibrium (though not conformational equilibrium) in one day. The volume occupied by a base pair as the DNA is hydrated (obtained from the x‐ray and swelling data) shows anomalies for the case of Na‐DNA in the region where the A‐form occurs. Our Raman and x‐ray data reproduce the well‐known features of the established conformational transitions, but we find evidence in the Raman spectra and optical properties of a transition to what may be a disordered B‐like conformation in Na‐DNA below 40% relative humidity. We have studied the effects of crystallinity on the A to B transition. We find that the transition to the B‐form is impeded in highly crystalline samples. In most samples, the transition occurs in three days (after putting the sample at 92% relative humidity) but in highly crystalline samples, the transition may take months. By comparing the high‐frequency Raman spectra of highly ordered and disordered films, we show that the extent of crystallinity controls the amount of A‐DNA formed when ethanol is used to dehydrate the films. We show that rapid dehydration (by laser heating) does not result in a B to A transition. A fiber that gives A‐type x‐ray reflections probably contains B‐like material in noncrystalline regions. The low‐frequency Raman spectrum is dominated by a band at about 25 cm−1 in both Na‐ and Li‐DNA. Another band is seen near 35 cm−1 in Na‐DNA at humidities where the sample is in the A‐form. In contrast to earlier reports, we find that the Raman intensity does not depend on fiber orientation relative to the scattering vector. The “35‐cm−1” band is largely depolarized (i.e. vertical polarization incident and horizontal polarization scattered, VH, or vice versa, HV) while the “25‐cm−1” band appears in both VV, VH and HV polarizations. These bands are all weaker in HH polarization. The “25‐cm−1” band may be due to a shearing motion of the phosphates and their associated counterions, while the “35‐cm−1” band may be characteristic of A‐DNA crystallites. We consider mass‐loading, relaxational coupling to the hydration shell, and softening of interatomic potentials as possible explanations of the observed softening of the low‐frequency Raman bands on hydration. Relaxation data suggest that the added water binds tightly (on these time scales) and a mass‐loading model accounts for the observed softening rather well.

We conclude that the A to B transition is not driven by softening of the “25‐cm−1” band. Rather, it is most probably a consequence of crystal‐packing forces, with the more regular A‐form favored in crystals when these forces are strong.
CD of ethidium bromide complexes with normal and electrophoretically anomalous DNA restriction fragments
Authors: Slobodyansky Elizabeth , Stellwagen John , Stellwagen Nancy C.
Published: December 2004

The equilibrium binding of ethidium bromide (EB) to two small 147 base‐pair (bp) DNA restriction fragments, which exhibit different mobilities in polyacrylamide gels, was investigated by CD. Two larger DNA restriction fragments and calf thymus DNA were also studied for comparison. Difference spectra were calculated by subtracting the spectrum of the pure DNA from the spectra of its DNA–EB complexes. The D/P ratios ranged from 0.03 to 1.0. The difference CD spectra of all fragments are characterized by bands with maxima near 310, 275, and 207 nm, and minima near 290, 253, 225, and 190 nm. The band near 310 nm, which has a shoulder at about 335 nm, has zero intensity at D/P ≤ 0.05, and rises to a plateau value, different for each fragment, at D/P ≃ 0.3 for large fragments (≥ 1400 bp), and D/P ∼ 0.7 for the two small 147 bp fragments. The minimum near 290 nm is markedly blue shifted with increasing D/P, the wavelength of the extremum corresponding approximately to the wavelength of the uv absorption maximum of the DNA–EB complex. The negative amplitude of this band at D/P = 1.0 depends on the molecular weight of the DNA. The difference CD maximum near 275 nm is positive at low D/P ratios, increases and goes through a maximum at D/P = 0.06–0.1, and then becomes increasingly negative with increasing D/P. The amplitude of the negative ellipticity per added dye is constant at high D/P ratios, suggesting that the transition can be attributed to outside‐bound EB molecules. The ellipticities at 310, 290, and 253 nm increase in absolute magnitude with increasing D/P at approximately the same rate, suggesting that all three bands are associated with the same optical and/or conformational transition. For the two small 147 bp fragments the fractional increases in amplitude of these bands parallel the fractional increase in length of the DNA upon binding EB, determined by electric birefringence measurements. The titration of the restriction fragments with EB was also followed by optical absorption. Two end points are observed, the first at a D/P ratio of ∼ 0.1, reflecting the transition between intercalated and outside‐bound dye molecules, and the second at D/P ≃ 1.0, the equivalence point of the titration.
A revisit to the three‐dimensional structure of B‐type starch
Authors: Imberty Anne , Perez Serge
Published: December 2004

A new three‐dimensional structure of B‐starch is proposed in which the unit cell contains 12 glucose residues located in two left‐handed, parallel‐stranded double helices packed in a parallel register; 36 water molecules are located between these helices. Chains are crystallized in the hexagonal space group P61, with lattice parameters a = b = 1.85 nm, c = 1.04 nm. The space group symmetry was derived from an exhaustive analysis of the large body of structural studies published so far. Diffraction data used in this work were taken from the previously reported x‐ray fiber diffractogram [H.C. Wu and A. Sarko (1978), Carbohydrate Research, 61, 7–25] after adequate reindexing. The final R factor is 0.145 for the three‐dimensional data. The repeating unit consists of a maltose molecule where the glucose residues have the 4C1 pyranose conformation and are α(1 → 4) linked. The conformation of the glycosidic linkage is characterized by torsion angles (Φ, Ψ) that take the values (83.8°, −144.6°) and (84.3°, −144.1°), whereas the valence angles at the glycosidic bridge have a magnitude of 115.8° and 116.5°, respectively. The primary hydroxyl groups exist in a gauche–gauche conformation. There is no intramolecular hydrogen bond. Within the double helix, interstrand stabilization is achieved without any steric conflict and through the occurrence of O(2)…O(6) type of hydrogen bonds. The model presented here, with an hydration around 27% w/w, corresponds to a well‐ordered crystalline sample, since all the water molecules could be located with no apparent sign of a disorder. Half of the water molecules are tightly bound to the double helices; the remainder forms a complex network centered around the sixfold screw axis of the unit cell. The consistency of the present structural model, with both physicochemical and biochemical aspects of the crystalline component of tuber starch granules, is analyzed.
On the multiple‐minima problem in the conformational analysis of polypeptides. II. An electrostatically driven Monte Carlo method—tests on poly(L‐alanine)
Authors: Ripoll Daniel R. , Scheraga Harold A.
Published: December 2004

A new approach to the multiple‐minima problem in protein folding is presented. It is assumed that the molecule is driven toward the native structure by three types of mechanism. The first one involves an optimization of the electrostatic interactions, whereby the molecule evolves toward conformations in which the charge distribution becomes energetically more favorable. The second mechanism involves a Monte Carlo–energy minimization approach, and the third one is a backtrack mechanism that acts in the opposite direction, increasing the energy—the third type of movement provides a means to perturb the molecule when it is trapped in a stable but energetically unfavorable local energy minimum. This paper describes the implementation of a model based on these mechanisms, and illustrates its effectiveness by computations on different arbitrary starting conformations of a terminally blocked 19‐residue chain of poly(L‐alanine) for which the global minimum apparently corresponds to the right‐handed α‐helix. In all cases, the global minimum was attained, even when the starting conformation was a left‐handed α‐helix. In the latter case, the trajectory of conformations passed through partially melted forms of the left‐handed α‐helix (because of electrostatic defects at the ends), and then through the formation of structures leading to the more stable right‐handed α‐helix.
Direct product‐matrix method treatment of macromolecular binding
Authors: Woodbury Charles P.
Published: December 2004

A major limitation of the well‐known matrix method treatment of linear polymer systems is the large size of the transfer matrix required to account simultaneously for such factors as polymer conformation, polymer sequence, ligand binding, etc. This paper shows how to combine such factors by using a direct product of small matrices, one for each factor, to generate the large transfer matrix. The properties of the direct product operation can be used to simplify considerably the calculation of eigenvalues and eigenvectors of the transfer matrix. This permits one to develop fairly simply the statistical thermodynamics of relatively complicated macromolecular binding systems involving a large number of states for the individual polymer sites. Applications include treatments of sequence‐specific binding of ligands, sequence‐specific conformational changes, and the combination of the two processes.
Micellization and interactions with phospholipid vesicles of the lipopeptide iturin a, as monitored by time‐resolved fluorescence of a D‐tyrosyl residue
Authors: Harnois I. , Genest D. , Brochon J. C. , Ptak M.
Published: December 2004

The micellization and the interactions with lipid vesicles of the antifungal cyclic lipopeptide iturin A have been investigated by nanosecond pulse fluorometry of a D‐tyrosyl residue. We show that this lipopeptide has three conformers in solution whose proportions are modified during the micellization process. Below the critical micellar concentration (CMC) iturin A does not self‐associate inside the bilayer. Above the CMC all the molecules of iturin A interact with the vesicles and self‐associate inside the membrane.
Order–Disorder conformation change of xanthan in 0.01M aqueous sodium chloride: Dimensional behavior
Authors: Liu Wei , Norisuye Takashi
Published: December 2004

Weight‐average molecular weights Mw, second virial coefficients, and z‐average radii of gyration 〈S2〉 were determined by light scattering as a function of temperature T for four sodium salt samples of xanthan in 0.01M aqueous NaCl, in which the polysaccharide undergoes an order–disorder conformation change with increasing T. The data for 〈S2〉 and Mw at 25 and 80°C, the lowest and highest temperatures studied, confirmed the previous conclusion that the predominant conformation at the former T, i.e., in the ordered state, is a double helix, while that at the latter T, i.e., in the disordered state, is a dimerized coil expanded by electrostatic repulsions between charged groups of the polymer. As T was increased from 25 to 80°C, 〈S2〉 sigmoidally decreased or increased depending on the dimer's molecular weight. This temperature dependence of 〈S2〉 and that determined elsewhere for a high molecular weight sample were found to be described almost quantitatively by a simple dimer model in which the double helix melts from both ends, when the double‐helical fraction in the dimer at a given T estimated previously from optical rotation data was used.
Chromatography and electron microscopy of cross‐linked fibrin polymers—a new model describing the cross‐linking at the DD‐trans contact of the fibrin molecules
Authors: Selmayr E. , Deffner M. , Bachmann L. , Müller‐Berghaus G.
Published: December 2004

Polymerization of fibrin molecules results in the formation of a double‐stranded protofibril. Although convincing data have not been presented, it is classically believed that γ‐chain cross‐linking of fibrin molecules occurs between the longitudinal end‐to‐end contacts (DD‐long contacts) of the molecules within each of the two strands of a protofibril (intrastrand cross‐linking). In this investigation the question addressed was whether γ‐chain cross‐linking takes place across the two strands (interstrand cross‐linking) between the transversal half‐staggered contacts of the molecules. Demonstration of double‐stranded protofibrils in the presence of urea would indicate an interstrand cross‐linking, whereas in the case of intrastrand cross‐linking, the chaotropic agent urea would dissociate the double‐stranded structure to form single‐stranded fibrils. Protofibrils were obtained by generating soluble cross‐linked fibrin polymers (sXLFbP): After incubation of souble fibrin polymers with Factor XIIIa at 37°C, the polymerization and cross‐linking reaction was stopped by the addition of 6M urea and EDTA. Gel filtration of the reaction mixture in the presence of 3M urea was effect in separating sXLFbP from monomeric molecules. The sXLFbP‐containing fractions were adsorbed onto mica in the presence of different concentrations of urea and investigated by electron microscopy after rotary shadowing. In the presence of 3M urea the sXLFbP appeared as double‐stranded protofibrils. In the presence of 4M urea some parts of the double‐stranded structure were found to be unfolded whereas in the presence of 6M urea multiple‐bended single‐stranded fibrils were observed. SDS‐polyacrylamide gel electrophoresis of the sXLFbP demonstrated no γ‐chain cross‐linking within the protofibrils. Ultracentrifugation of the sXLFbP showed that in the presence of 3M urea noncross‐linked fibrin polymers dissociated to monomeric molecules. When sXLFbP was centrifuged into 6M urea on sucrose density gradients, no reduction of the polymer size could be observed. The data indicate that γ‐chain cross‐linking occurs between the transversal contacts of the fibrin molecules within a protofibril, thus generating interstrand cross‐linking. A model of the cross‐linking of polymerized fibrin molecules is developed and the term DD‐trans contact is proposed for this specific alignment of the D‐domains.
Helix–Coil transition of plasmid dna by adiabatic differential scanning microcalorimetry
Authors: Maeda Y. , Takahashi K. , Yamaki H. , Ohtsubo E.
Published: December 2004

Adiabatic differential scanning microcalorimetry, which provides curves of the heat capacity vs temperature, was carried out for the DNA of plasmid pJL3‐TB5 (5277 base pairs in length). The calorimetry curve shows nine peaks ranging from 81 to 96°C in 1 × SSC buffer at a heating rate of 0.25°C, due to the stepwise helix–coil transition of the DNA along the molecular chain. The theoretical melting curve, which can be constructed by calculation from the entire nucleotide sequence of the plasmid DNA by the helix–coil transition theory, is then compared with the calorimetry curve. The two curves resemble each other remarkably well, particularly when a parameter for the methylated adenine residues at GATC sites by Dam methylase is used appropriately. This allows us to assign each peak in the calorimetry curve to the melting of the respective regions of the plasmid DNA sequence. The local stability of the helix–coil transition along the DNA chain is closely related to the functional regions coded by pJL3‐TB5, such as genes, transcriptional promoters, and particular sites generated by recombination of two different sequences in vivo and in vitro.