bluuebunny/crossref_metadata_2025_split huggingface dataset:
Orientational dynamics of T2 DNA during agarose gel electrophoresis: Influence of gel concentration and electric field strength
Authors: Åkerman Björn , Jonsson Mats , Nordén Bengt , Lalande Marc 
Published: December 2004

AbstractThe understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration (“electrophoretic orientation”) is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Åkerman, and B. Nordén, (1988) Biopolymers 27, 381–414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23‐kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3–2%), electric field strength (0–40 V/cm), and pulse characteristics.The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady‐state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over‐ and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented.The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.
On the cooperative binding of large ligands to a one‐dimensional homogeneous lattice: The generalized three‐state lattice model
Authors: Bujalowski Wlodzimierz , Lohman Timothy M. , Anderson Charles F. 
Published: December 2004

AbstractWe present the general secular equation for three‐state lattice models for the cooperative binding of large ligands to a one‐dimensional lattice. In addition, a closed‐form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter ω(0 < ω < ∞) thus eliminating the need for multiple equations.
Protein complexation with DNA phosphates as a cause for DNA duplex destabilization: A thermodynamic model
Authors: Van Genderen Marcel H. P. , Buck Henk M. 
Published: December 2004

AbstractComplexation of positively charged sites in a protein with the negative DNA phosphate groups shields the phosphate charges. This diminishes interstrand electrostatic repulsions, which stabilizes the duplex. When phosphate shidlding is present in one DNA strand only, the conformation of this strand changes due to a decrease of intrastrand phosphate–phosphate repulsions. This destabilizes the duplex since then the strands differ in conformation. A thermodynamic model is formulated to describe this stabilization/destabilization effect in terms of changed enthalpies and entropies of hybridization. It is found that protein complexation with one DNA strand can indeed lower the TM value of a duplex. The model is applied to the action of helicases (replication), RNA polymerases (transcription), and restriction endonucleases. Mechanisms with unilateral charge shielding are proposed for their duplex‐destabilizing properties.
Theory of cooperative transitions in protein molecules. II. Phase diagram for a protein molecule in solution
Authors: Finkelstein Alexey V. , Shakhnovich Evgeny I. 
Published: December 2004

AbstractThe thermodynamically stable states of denatured protein in solution are investigated. These states are distinguished from the native state by the absence of tight packing of side chains while the compactness of denatured protein may vary within a wide region. The following regimes are outlined:

the “wet” molten globule, i.e., the compact state with pores occupied by solvent;
the swollen globule (“wet,” of course); and
the coil.
The “dry” molten globule, when solvent does not penetrate inside the protein, is excluded for all experimental conditions. All the transitions within the denatured globule state are gradual while the denatured globule–coil phase transition is a second order one. The conditions of protein denaturation as well as conditions of transitions and crossovers within the denatured state are outlined.
Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization
Authors: Robert Charles H. , Colosimo Alfredo , Gill Stanley J. 
Published: December 2004

AbstractWe examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1–14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.
IR vibrational CD in alanyl tripeptide: Indication of a stable solution conformer
Authors: Lee O. , Roberts G. M. , Diem M. 
Published: December 2004

AbstractInfrared vibrational CD (VCD) of a small peptide, L‐alanyl‐L‐alanyl‐L‐alanine (Ala3), and a peptide model, N‐acetyl‐L‐alanine‐N′‐methyl‐amide (AAMA), in the 1550–1750‐cm−1 region has been observed. The “coupled oscillator” VCD feature observed for Ala3 in the amide I region is interpreted in terms of a solution structure stabilized by the electrostatic interaction of the zwitterionic groups. No such interactions are possible in basic aqueous solution of Ala3 nor in AAMA in neutral solution. Thus, the coupled oscillator features are lost in the latter two cases, indicating the absence of a simple stabilized conformation.
A critical examination of the reaction of pyridoxal 5‐phosphate with human hemoglobin Ao
Authors: Marini Mario A. , Moore Gerald L. , Fishman Ronald M. , Jessee Robert , Medina Frank , Snell Shawn M. , Zegna Angelo I. 
Published: December 2004

AbstractPyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 ± 2 torr and a Hill coefficient of 2.3 ± 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen‐ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., > 10%). Exessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with < 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5‐phosphate (PLP) shows three major bands for the cyanoborohydride‐reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed‐bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 ± 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4°C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse‐phase chromatography indicates that the addition of PLP to HbAo is directed solely to the β‐chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the α‐ and β‐chains.
Differential scanning calorimetric study of the thermal unfolding of myosin rod, light meromyosin, and subfragment 2
Authors: Lopez‐Lacomba Jose L. , Guzman M. , Cortijo Manuel , Mateo Pedro L. , Aguirre Raul , Harvey Stephen C. , Cheung Herbert C. 
Published: December 2004

AbstractThe thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S‐2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G‐200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation‐renaturation followed by Sephacryl S‐200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two‐state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S‐2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S‐2, but absent in the DSC curve for LMM. This transition was identified with the high‐temperature transition detected with long S‐2 and attributed to the melting of the coiled‐coil α‐helical segment of subfragment 2 (short S‐2). The low‐temperature transition of long S‐2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C‐terminal segment of long S‐2, and a short N‐terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S‐2/LMM junction, and upon proteolytic cleavage yields the C‐terminal and N‐terminal ends of long S‐2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S‐2. This finding provides an additional argument for the existence of independent domains in myosin rod.
Autoxidation of ascorbic acid catalyzed by a semisynthetic enzyme
Authors: Germanas Juris P. , Kaiser Emil T. 
Published: December 2004

AbstractThe semisyntehtic enzyme 6 was prepared by alkylation of the cysteine‐25 sulfhydryl group of papain with the bipyridine 5 and was shown to stoichiometrically bind copper ion; 7 catalyzed the autoxidation of ascorbic acid derivatives with saturation kinetics approximately 20‐fold faster than a model system using 3‐Cu(II).
Structure and conformation of the nitroxyl spin‐label ethyl 3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oate determined by electron nuclear double resonance: Comparison with the structure of a spin‐label substrate of carboxypeptidase A
Authors: Mustafi Devkumar , Boisvert William E. , Makinen Marvin W. 
Published: December 2004

AbstractThe conformation of the nitroxyl spin‐label ethyl 3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oate has been determined by electron nuclear double resonance (ENDOR) spectroscopy and computer‐based molecular modeling. From ENDOR spectra of the compound in frozen solution, we have assigned resonance absorption features for each class of protons, and we have identified their principal hyperfine coupling (hfc) components from analysis of the dependence of ENDOR spectra on the static laboratory magnetic field. The dipolar hfc components yielded estimates of the electron‐proton separations for each class of protons of the ethyl propenoyl moeity. Torsion angle search calculations were carried out to determine the conformational space compatible with hard‐sphere nonbonded constraints and with the ENDOR‐determined distance constraints. Molecular graphics analysis revealed that the propenoyl side chain of the spin‐label exhibits an extended trans conformation and that the ethyl moiety of the ester group deviates significantly from coplanarity with the carboxylate COO atoms. The conformation of this molecule is compared with that of an analogous compound O‐[3‐(2,2,5,5‐tetramethylpyrrolinyl‐1‐oxyl)‐propen‐2‐oyl]‐L‐β‐phenyllactate, which has been employed as a spectroscopic substrate probe of carboxypeptidase A (L. C. Kuo, J. M. Fukuyama, and M. W. Makinen (1983) Journal of Molecular Biology 163, 63–105). The rotamer conformation of the free spin‐label ester in solution, as determined in this study, and that of the enzyme‐bound spin‐labeled phenyllactate are compared. Differences in rotamer structure are discussed in terms of stereoelectronic principles that govern the pathway of substrate hydrolysis catalyzed by carboxypeptidase A.
Inactivation of the catalytic subunit of bovine cAMP‐dependent protein kinase by a peptide‐based affinity inactivator
Authors: Mobashery Shahriar , Doughty Michael , Kaiser E. T. 
Published: December 2004

AbstractA peptide affinity inactivator, Ac‐Leu‐Arg‐Arg‐Ala‐(BrAc)Orn‐Leu‐Gly, was used as a tool to probe for active site residues in the catalytic subunit of bovine cAMP‐dependent protein kinase. The peptide inactivated the catalytic subunit in an active site‐directed and monophasic manner with a first‐order rate constant of 0.03 min−1 and a dissociation constant of 675 μM. Studies with radioactive peptide indicated that approximately one equivalent of peptide was incorporated into each protein molecule. Protein sequencing identified the modified residue as Cys‐199. A possible location for Cys‐199 within the active site is suggested.
Construction, expression, and preliminary characterization of chimeric class μ glutathione S‐transferases with altered catalytic properties
Authors: Zhang Pinghui , Armstrong Richard N. 
Published: December 2004

AbstractAn expression plasmid for isoenzyme 3–3 of rat liver glutathione S‐transferase has been constructed from the cDNA clone pGTA/C44 and the pAS expression vector pMG27NS, and used for the efficient production of the enzyme in the Escherichia coli strain M5219. The plasmid has also been manipulated, through theuse of synthetic linkers, to encode chimeric polypeptides in which short sequnces of the closely related isoenzyme 4–4 have been substituted into the N‐terminal and C‐terminal variable domains of isoenzyme 3–3. The chimeric polypeptides designated 493208, 320948, and 49320048 are expressed with varying degrees of efficiency in E. coli. The active dimeric holoenzymes 3‐3, (493208)2, (320948)2, and (49320048)2 can be isolated. The spectroscopic and kinetic properties of the chimeric enzymes are significantly different than the native enzyme.
Light scattering studies of the effect of Ca2+ on the structure of porcine submaxillary mucin
Authors: Varma B. K. , Demers A. , Jamieson A. M. , Blackwell J. , Jentoft Neil 
Published: December 2004

AbstractThe effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCl2: these data are within experimental error of those for solutions in 0.1M NaCl. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z‐average translational diffusion coefficient and the longest intramolecular relaxation time τ1. A plot of τ1 vs 〈1/Rh〉 is linear, and consistent with plots of such data recorded for PSM in 0.1M NaCl and 6M GdnHCl solutions. However, the τ1 values and the associated results for 〈R〉 in 0.5M CaCl2 are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCl2 solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.
Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: Effect of alkylamine chains
Authors: Hopkins Harry P. , Fumero Juan , Wilson W. David 
Published: December 2004

AbstractCalorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15–60°C range. Enthalpy changes (ΔHB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of ±0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), ΔHB varies little with temperature, and the heat capacity change (ΔCP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, ΔHB changes from −6.5 to −8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and ΔCP is −57 cal/deg/mol. At 21°C a plot of ΔHB vs mole ratio is curved downward for propidium in the 0.08–0.25 range, whereas the same plot at 45°C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50°C. These observations and our analyses of ΔHB and ΔSB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50°C is ΔHB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for ΔHB is not available.
A unique binding cavity for divalent cations in the DNA–metal–chromomycin A3 complex
Authors: Itzhaki Laura , Weinberger Sarah , Livnah Nurit , Berman Elisha 
Published: December 2004

AbstractBinding of chromomycin A3 (CRA) to calf thymas DNA was investigated in the presence of divalent cations using visible absorption and 1H‐nmr spectroscopies. An apparent equilibrium binding constant (∼ 1011M−1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA–M–CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H‐nmr spectrum of the above complex using the paramagnetic Co2+ cation indicating that the metal occupies a unique binding site. Since no induced 1H‐nmr chemical shifts were observed for the drug–Co2+ mixture, it was concluded that no metal–drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (< 0.85 Å) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA–M–CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.
Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels
Authors: Wu Chi , Wang Zhulun , Chu Benjamin 
Published: December 2004

AbstractBy combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed‐field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.
Simulations of the B‐DNA molecular dynamics of d(CGCGAATTCGCG)2 and d(GCGCGCGCGC)2: An analysis of the role of initial geometry and a comparison of united and all‐atom models
Authors: Rao Shashidhar N. , Kollman Peter 
Published: December 2004

AbstractMolecular dynamics simulations on the sequence d(CGCGAATTCGCG)2 have been carried out using both united atom and all‐atom representations, and starting the simulations both from a regular repeating B‐DNA structure and from the x‐ray single crystal B‐DNA structure. An all‐atom B‐DNA simulation on the sequence d(GCGCGCGCGC)2 has also been carried out, in order to compare it with a previous united atom simulation. The helix repeats, H‐bonding, sugar pucker profiles, and average torsional angles are all in the range observed in crystallographic and nmr studies for B‐DNA helices. In some of the sequences, there is a significant bend in the DNA helices. The individual helix repeats, with focus on 3′CpG5′ and 3′GpC5′ units, show the opposite helix repeat to that suggested by Calladine's rules.