Molecular dynamics simulation study of probe diffusion in liquid n-alkanes

We performed molecular dynamics simulations for the probe diffusion and friction dynamics of Lennard-Jones (LJ) particles modelled for methyl yellow (MY) in liquid n-alkanes of C₁₂–C₂₀₀ at temperatures of 318, 418, 518 and 618 K. Two LJ particles are chosen: MY1 with a mass of 114 g/mol, LJ parameters of σ = 4.0 Å and ? = 0.4 kJ/mol, and MY2 with a mass of 225 g/mol, σ = 6.0 Å and ? = 0.6 kJ/mol. We observed a clear transition in the power law dependence of MY2 diffusion on the molecular weight of n-alkanes at lower temperatures of 318 and 418 K. The sharp transitions occur near n-dotriacontane (C₃₂). However, no such transition is found for MY1 at all the temperatures and for MY2 at higher temperatures of 518 and 618 K. We also calculated the friction constants of both MY probe molecules in liquid n-alkanes. For the larger probe molecule (MY2), at lower temperatures, a large deviation of slope from the linear dependence of the friction of MY2 on the chain length of n-alkane is observed, which indicates a large reduction of friction in longer chains when compared with the shorter chains, enhancing the diffusion of the probe molecules (MY2).     

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific ¹³C/¹⁵N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers’ spatial and temporal resolution down to today’s values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.     

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers’ spatial and temporal resolution down to today’s values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.     

Study on properties of liquid ammonia via molecular dynamics simulation based on ABEEMσπ polarisable force field

Abstract

The molecular dynamics simulation has been performed to investigate the charge distribution, structural and dynamical properties of liquid ammonia at 273 K using a polarisable force field of the atom-bond electronegativity equalisation method (ABEEMσπ). One ammonia molecule in this model has eight charge sites, one N atomic site, three H atomic sites, three N–H bond sites and one lone-pair electron site. ABEEMσπ model can present the quantitative site charges of molecular ammonias in liquid and their changing in response to their surroundings. The radial distribution functions and dynamical properties are in fair agreement with the available experimental data. The first peak of g_NN(r) appears at N–N distance of ~3.50 ± 0.05 Å where most hydrogen bonds are formed. The average coordination number of the first shell is 13.0 ± 0.1 among which a central ammonia molecule intimately connects 3 ~ 4 ammonia molecules by hydrogen bonds. The power spectrum shows the vibrations of hydrogen bonds. For a reference, a simple estimation of the average hydrogen bonding energy in liquid ammonia is 6.5 ± 0.1 kcal/mol larger than 3.8 ± 0.3 kcal/mol in dimer ammonia. Our simulation results provide more detailed information about liquid ammonia.     

DNA-binding study of anticancer drug cytarabine by spectroscopic and molecular docking techniques

The interaction of anticancer drug cytarabine with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multispectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove-binding mode, while the binding constant of UV-vis and the number of binding sites were 4.0 ± 0.2 × 10⁴ L mol^?1 and 1.39, respectively. The fluorimetric studies showed that the reaction between the drugs with CT-DNA is exothermic. Circular dichroism spectroscopy was employed to measure the conformational change of DNA in the presence of cytarabine. Furthermore, the drug induces detectable changes in its viscosity for DNA interaction. The molecular modeling results illustrated that cytarabine strongly binds to groove of DNA by relative binding energy of docked structure ?20.61 KJ mol^?1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the interaction of small molecular pollutants and drugs with biomacromolecules for clarifying the molecular mechanism of toxicity or side effect in vivo.     

Measuring fast dynamics in solutions and cells with a laser scanning microscope

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.     

Binding of glycoproteins of microsomal and Golgi membranes to lectins.

Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of $\text{Torpedo ocellata}$, have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with $\text{p}$-($\text{N}$-maleimido)benzyl [trimethyl-³H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (K_Tb ? 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (K_Ca ? 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.     

Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine–DNA adduct formation

Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine–DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine–DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time–drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (K_a) for semustine and lomustine interactions with DNA are 1.53?×?10³ M^?1 and 8.12?×?10³ M^?1, respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

DNA Electrochemical Sensor for Detection of PRSS1 Point Mutation Based on Restriction Endonuclease Technique

To construct a restriction endonuclease based biosensor technology for PRSS1 genotyping. We designed a thiol-modified hairpin probe where the neck has EcoRI endonuclease recognition sites according to the PRSS1 gene c.410 C>T (p.T137 M) mutation and it was fixed on the gold electrode. Different charge generated by the binding of MB to phosphate groups of DNA before and after hybridization was used for distinguishing the different genotypes and quantity. This showed that the novel sensor can better distinguish the complementary sequence, single-base mismatches, and completely noncomplementary sequences, and the linear range for the logarithm was Y = –0.0242 X + 0.1574, R = 0.9912(Y = current, X = log target DNA concentration); the detection limit for DNA detection is estimated to be 50 fM.     

QSAR-based prediction of anti-HCV activity of thiourea derivatives

Quantitative structure–activity relationship (QSAR) studies were performed on a series of thioureas to explore the physico-chemical parameters responsible for their activity against the hepatitis C virus (HCV)-infected AVa5 cell. The physico-chemical parameters were calculated using WIN CAChe 6.1. Multiple linear regression analysis, after the variables selection by factor analysis, was performed to derive QSAR models which were further evaluated for their statistical significance and predictive power by internal and external validation. The developed QSAR model had the correlation coefficient (R) = 0.928 and cross-validated squared correlation coefficient (Q ²) = 0.751. The selected significant QSAR model indicates that hydrophobicity, dielectric energy, valence connectivity index (order 1), conformational minimum energy and highest occupied molecular orbital of the whole molecule play an important role in the anti-HCV activity of thioureas.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.     

Real-time observation of a single DNA digestion by λ exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA–protein interactions, such as the DNA digestion reaction by λ exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual λ exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of λ exonuclease, a stained λ DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by λ exonuclease revealed that the DNA digestion rate was ~1000 bases/s and also demonstrated high processivity.     

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhA_C, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.     

Flow cytometric analysis in Lagenaria siceraria (Cucurbitaceae) indicates correlation of genome size with usage types and growing elevation

The occurrence and extent of genome size variation within species is controversially discussed and thorough analyses are rare. Given the large morphological variation in Lagenaria siceraria (bottle gourds) and its wide distribution in Africa we here analysed (1) the genome size variation within cultivars of L. siceraria, (2) the correlation between genome size and morphological traits, and (3) the geographical patterns of DNA content within the species. We measured 2C-values of 366 individuals from 117 accessions of L. siceraria (2n = 22) from Africa, America and Asia via flow cytometry with propidium iodide as DNA stain. We found that 2C-value in L. siceraria (0.683–0.776 pg/2C) is about two times lower than previously reported and varies by about 12% among all accessions. Moreover, our results indicated a clear correlation of genome size with two different seed or usage types and with growing elevations in West Africa. Within the seed types genome size varies by 6.6 and 7.5%, respectively. The genome size differences in seed types of L. siceraria might indicate differences in their evolutionary history and necessitates a re-evaluation of the phylogenetic relationships within L. siceraria while the correlation between 2C-value and the elevation of the collecting sites might indicate an adaptation of genome size to an unknown ecological parameter connected to altitude.