Temperature dependence of the thermodynamics of helix-coil transition

The temperature-induced helix to coil transition in a series of host peptides was monitored using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). Combination of these two techniques allowed direct determination of the enthalpy of helix-coil transition for the studied peptides. It was found that the enthalpy of the helix-coil transition differs for different peptides and this difference is related to the difference in the temperature for the midpoint of helix-coil transition. The enthalpy of the helix-coil transition decreases with the increase in temperature, thus providing the first experimental estimate for the heat capacity changes upon helix-coil transition, DeltaC(p). The values for DeltaC(p) of helix-coil transition are found to be negative, which is in contrast to the positive DeltaC(p) for protein unfolding. Analysis suggests that this negative DeltaC(p) of helix-coil transition is due to the exposure of the polar peptide backbone to solvent upon helix unfolding.     

Nonsequence-specific arginine interactions in the nucleosome core particle

We have analyzed the relative orientation of basic amino acid side chains towards DNA in the nucleosome core particle. The electric field created by DNA phosphates has no apparent preferential orientation: no favored orientation of the arginine guanidinium group is found. Arginine may be either directly hydrogen bonded to a phosphate oxygen or stabilized in the minor groove by van der Waals contacts and the local negative electric field. On the other hand, the phosphate oxygen atoms hydrogen bonded to arginines are always found close to the plane defined by the guanidinium group. Thus it can be concluded that the interactions of arginine are strongly directional, those of phosphate are not. We also find that a highly charged fragment of histone H2B, which is placed between two DNA turns, has a very variable conformation. An increase in protein positive charge density apparently allows multiple nonspecific protein conformations when interacting with DNA.     

DNA immunization with fusion genes containing HCV core region and HBV core region

The eucaryotic expression plasmids were constructed to express the complete (HCc191) or the truncated (HCc69 and HCc40) HCV core genes, solely or fused with the HBV core gene (HBc144). These constructions were transiently expressed in COS cells under the control of the CMV promoter. The antigenicity of HBc and HCc could be detected in the expression products by ELISA and Western blot. The mice immunized with these expression plasmids efficiently produced the anti-HCc antibodies, and also anti-HBc antibodies when the plasmids contained the fusion genes. In addition, the antibodies induced by the fusion genes were more persistent than those induced by the non-fusion HCV core genes. These indicate that the fusion of HCc genes to HBc gene is in favor of the immunogenicity of HCc, while the immunogenicity of HBc is not affected.     

DNA stretching and extreme kinking in the nucleosome core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.     

Redefinition of the cleavage sites of DNase I on the nucleosome core particle

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.     

Effects of Temperature,pH and Salt Concentrations on the Adsorption of Bacillus subtilis on Soil Clay Minerals Investigated by Microcalorimetry

Adsorption of microorganisms on minerals is a ubiquitous interfacial phenomenon in soil. Knowledge of the extent and mechanisms of bacterial adsorption on minerals is of great agronomic and environmental importance. This study examined adsorption of Bacillus subtilis on three common minerals in soils such as kaolinite, montmorillonite and goethite under various environmental conditions. Isothermal titration calorimetry (ITC) was used to investigate the effects of temperature (20, 30, and 40°C), pH (5.0, 7.0, and 9.0) and KNO₃ concentration (0.001, 0.01, and 0.1 mol L^?1) on the adsorption by direct measurement of enthalpies. The results revealed that the adsorption process in all the mineral systems were exothermic, with the enthalpy changes (ΔH_ads ) ranging from ?52 to ?137, ?33 to ?147, and ?53 to ?141 kJ kg^?1 (dry weight of adsorbed bacteria) for kaolinite, montmorillonite, and goethite, respectively. No obvious dependence of ΔH_ads on temperature was observed. The heat release for all the systems generally declined with pH and decrease of salt concentration, which can be explained by the variations of hydrophobicity and electrostatic force with pH or salt concentration. The largest decrease was found for goethite among the three minerals from pH 5.0 to 7.0, suggesting that electrostatic attraction may play a more important role in bacterial adsorption on this mineral. The ΔH_ads values for all the minerals became nearly the same at pH 9.0, indicating that the same force probably hydrophobicity governing the adsorption for the minerals in alkaline environment. It is assumed that acidic or saline soils and the associated environments favor the adsorption of B. subtilis on clay minerals. In addition, the negative enthalpies expressed as kJ kg^?1 (carbon) revealed an energy flow into the environment accompanied by the carbon adsorption on the minerals in soil.     

Glass transition and enthalpy relaxation of amorphous lactose glass

The glass transition temperature, T(g), and enthalpy relaxation of amorphous lactose glass were investigated by differential scanning calorimetry (DSC) for isothermal aging periods at various temperatures (25, 60, 75, and 90 degrees C) below T(g). Both T(g) and enthalpy relaxation were found to increase with increasing aging time and temperature. The enthalpy relaxation increased approximately exponentially with aging time at a temperature (90 degrees C) close to T(g) (102 degrees C). There was no significant change observed in the enthalpy relaxation around room temperature (25 degrees C) over an aging period of 1month. The Kohlrausch-Williams-Watts (KWW) model was able to fit the experimental enthalpy relaxation data well. The relaxation distribution parameter (beta) was determined to be in the range 0.81-0.89. The enthalpy relaxation time constant (tau) increased with decreasing aging temperature. The observed enthalpy relaxation data showed that molecular mobility in amorphous lactose glass was higher at temperatures closer to T(g). Lactose glass was stable for a long time at 25 degrees C. These findings should be helpful for improving the processing and storage stability of amorphous lactose and lactose containing food and pharmaceutical products.     

Combining particle tracking microrheology and viscometry for the study of DNA aqueous solutions

We use video particle tracking microrheology (VPTMR) in order to investigate the viscoelasticity of salmon DNA and correlate it to its steady-flow shear-thinning viscosity. Aqueous solutions of DNA are tested in a wide concentration range from the dilute to the semidilute unentangled concentration regime. The observed mean squared displacement shows power-law scaling with lag-time which is equivalent to power-law behavior of the complex modulus as a function of frequency that is, |G^*(ω)| = S ∙ ω ^α . The relaxation exponent α changes abruptly with concentration in the semidilute regime from about 1 to about 0.5 which is the exponent predicted by the Rouse model. The quasi-property S follows the scaling of viscosity for uncharged polymers near θ-conditions in the semidilute regime that is, with ν_eff = 0.50 − 0.51 . The shear-thinning exponent observed by viscometry increases gradually towards the value of 0.5 which has been predicted for Rouse chains under flow. Our findings are in agreement with recent studies of DNA solutions where DNA is treated as a model polymer and addresses the low-molar mass regime of DNA viscoelasticity. This work demonstrates that the combination of passive particle tracking with viscometry can provide a complete picture on the viscoelasticity of DNA-based biopolymer materials.     

The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomic force microscopy

The structure of the nuclosome core particle of chromatin in chicken erythrocytes has been examined by using AFM.The 146 bp of DNA wrapped twice around the core histone octamer are clearly visualized.Both the ends of entry/exit of linker DNA are also demonstrated.The dimension of the nucleosome core particles is - 1-4 nm in height and - 13-22 nm in width.In addition,superbeads (width of - 48-57 nm,height of - 2-3 nm )are occasionally revealed,two turns of DNA around the core particles are also detected.     

Construction of an intermediate-resolution lattice model and re-examination of the helix-coil transition: a dynamic Monte Carlo simulation

In protein modeling, spatial resolution and computational efficiency are always incompatible. As a compromise, an intermediate-resolution lattice model has been constructed in the present work. Each residue is decomposed into four basic units, i.e. the α-carbon group, the carboxyl group, the imino group, and the side-chain group, and each basic coarse-grained unit is represented by a minimum cubic box with eight lattice sites. The spacing of the lattice is about 0.56?Å, holding the highest spatial resolution for the present lattice protein models. As the first report of this new model, the helix-coil transition of a polyalanine chain was examined via dynamic Monte Carlo simulation. The period of formed α-helix was about 3.68 residues, close to that of a natural α-helix. The resultant backbone motion was found to be in the realistic regions of the conformational space in the Ramachandran plot. Helix propagation constant and nucleation constant were further determined through the dynamic hydrogen bonding process and torsional angle variation, and the results were used to make comparison between classical Zimm-Bragg theory and Lifson-Roig theory based on the Qian-Schellman relationship. The simulation results confirmed that our lattice model can reproduce the helix-coil transition of polypeptide and construct a moderately fine α-helix conformation without significantly weakening the priority in efficiency for a lattice model.     

Thermodynamics of single strand DNA base stacking

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<-->U transition in this temperature range. The transition properties of the ssDNA sequences with 相似文献   

The structure of duplex DNA in the nucleosome core particle: An alternative model

Many studies indirectly indicate that the conformation ofin vivo duplex DNA is the double helix. The most direct view, from the X-ray analysis of the nucleosome core particle, has also been interpreted in terms of the double helix structure. However, an alternative possibility exists; that the duplex adopts a metastable side-by-side conformation which readily converts to the double helix on removal of protein. Evidence for the existence of this conformation has been obtained from a reanalysis of the electron density map for the nucleosome particle.     

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: effect of alkylamine chains

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) 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), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB 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 delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees 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 degrees C. These observations and our analyses of delta HB and delta SB 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 degrees C is delta HB 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 delta HB is not available.     

Multidimensional spectroscopic data correlation in the conformation transition of biological macromolecules

A computerized spectrophotometer system which is capable of simultaneously obtaining three spectral dimensions, that is, the absorbance, the circular dichroism and the fluorescence intensity, from one sample solution has been developed for the purpose of attaining a higher resolving power in the study of conformational transitions of biological macromolecules. Measurement conditions, such as the wavelength, the temperature, pH or the concentration of reagents in the sample solution, can be scanned according to a sequence that is set just prior to the measurement. A computer-driven micro-injector and a pH electrode directly immersed in the sample solution make it possible to obtain a titration curve in parallel to the optical measurement. All the data taken are stored on a magnetic disk for later retrieval. They can be processed and displayed in any required form. The helix-coil transitions of a polynucleotide caused by temperature and those of a polypeptide caused by pH, and the denaturation of proteins caused by guanidine hydrochloride, were studied by this measuring system. The continuous plotting of transition profiles and the correlation diagrams among different spectral dimensions has proved to be a good way of demonstrating the existence of different modes of transition.     

抗疟药物氯奎、阿的平、四氢-9-氨基(口了)啶与双链小牛胸腺DNA的结合

本文用体相微量量热计所得数据,估算了两种抗疟药物(antimalarial)分子和两种结构上相似的分子与DNA结合时的标准结合热焓。根据在不同离子强度下测定的表观结合常数获得了热力学结合常数。根据这些数据以及标准结合热焓计算出标准结合自由能、标准结合熵。结果发现凡与DNA结合的分子,具有线性芳环结构的,结合时主要取决于热焓的大小。具有脂肪链或脂肪环结构的,结合时主要取决于熵变。     

Restrained molecular dynamics of solvated duplex DNA using the particle mesh Ewald method

Restrained and unrestrained aqueous solution molecular dynamics simulations applying the particle mesh Ewald (PME) method to DNA duplex structures previously determined via in vacuo restrained molecular dynamics with NMR-derived restraints are reported. Without experimental restraints, the DNA decamer, d(CATTTGCATC)d(GATGCAAATG) and trisdecamer, d(AGCTTGCCTTGAG)d(CTCAAGGCAAGCT), structures are stable on the nanosecond time scale and adopt conformations in the B-DNA family. These free DNA simulations exhibit behavior characteristic of PME simulations previously performed on DNA sequences, including a low helical twist, frequent sugar pucker transitions, BI- BII(–) transitions and coupled crankshaft (–) motion. Refinement protocols similar to the original in vacuo restrained molecular dynamics (RMD) refinements but in aqueous solution using the Cornell et al. force field [Cornell et al. (1995) J. Am. Chem. Soc., 117, 5179–5197] and a particle mesh Ewald treatment produce structures which fit the restraints very well and are very similar to the original in vacuo NMR structure, except for a significant difference in the average helical twist. Figures of merit for the average structure found in the RMD PME decamer simulations in solution are equivalent to the original in vacuo NMR structure while the figures of merit for the free MD simulations are significantly higher. The free MD simulations with the PME method, however, lead to some sequence-dependent structural features in common with the NMR structures, unlike free MD calculations with earlier force fields and protocols. There is some suggestion that the improved handling of electrostatics by PME improves long-range structural aspects which are not well defined by the short-range nature of NMR restraints.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

Thermodynamics of the Binding of Chymotrypsin with the Black-eyed Pea Trypsin and Chymotrypsin Inhibitor (BTCI)

The binding of -chymotrypsin to black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) has been studied using the inhibitory activity against the enzyme and the formation of the complex enzyme/inhibitor followed by measurements of fluorescence polarization. Apparent equilibrium constants were estimated for several temperatures and the values obtained range from 0.32 × 10⁷ to 1.36 × 10⁷ M^–1. The following values were found from van't Hoff plots: H _vh ^° = 10.8 kcal mol^-1 (from inhibitory assays) and 11.1 kcal mol^–1 (from fluorescence polarization); S° = 67.9 and = 67.8 kcal K^–1 mol^–1, respectively. Calorimetric binding enthalpy was determined (corrected for the ionization heat of the buffer) and the resulting value was H _cal ^° = 4.9 kcal mol^-1. These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process.     

Effect of substitution of photo-cross-linker in photochemical cytosine to uracil transition in DNA

Genome editing is an important technique for protein engineering, treatment of genetic disorders, and production of non-native proteins. A shortcoming of current enzymatic and chemical methods for genome editing is their limited applicability for in vivo studies. In addition, non-enzymatic methods, such as photochemical DNA editing using 3-cyanovinylcarbazole (^CNVK), require high temperatures to affect cytosine to uracil transformations. To overcome this limitation, we developed new photo-cross-linkers based on ^CNVK, 3-methoxycarbonlycarbazole, 3-carboxyvinylcarbazole, and 3-carbonylamidevinylcarbazole. The use of 3-carboxyvinylcarbazole resulted in greater acceleration of the deamination reaction than that achieved with ^CNVK. The most likely factors affecting the ability of ultrafast photo-responsive nucleosides to accelerate the deamination reaction are polarity and hydrophilicity of the oligodeoxyribonucleotides that contain photo-cross-linker.