Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 cells.

Rat ascites hepatoma AH66 cells have lower sensitivity to Vinca alkaloids and anthracycline antibiotics than AH66F cells, a subline of AH66 cells. AH66 cells expressed P-glycoprotein, while the protein was not detectable in AH66F cells. There are two affinity sites for [3H]vinblastine binding in the AH66 cell membrane, while AH66F cells have only one affinity site. The high affinity [3H]vinblastine binding in AH66 cells was inhibited by Adriamycin, verapamil, nicardipine, and reserpine. The high affinity site of the binding may be the multidrug transporter, P-glycoprotein. [3H]Vinblastine binding was not influenced by adenosine 3'-5'-monophosphate (AMP), adenosine triphosphate (ATP), or guanosine triphosphate (GTP). The multidrug resistance in AH66 cells may depend on P-glycoprotein which is not modulated by nucleotide.     

Activation of tubulin assembly into microtubules upon a series of repeated femtosecond laser impulses

Tubulin, a globular protein, mostly distributed in nature in the dimeric alpha, beta form, can polymerize in vivo and in vitro into microtubules-longitudinal dynamic assemblies, involved in numerous cellular functions, including cell division and signaling. Tubulin polymerization starts upon binding Mg(2+) with the tubulin guanosine triphosphate (GTP) site. In the current study we show that a series of repeated femtosecond laser impulses activate the same site without adding Mg(2+). GTP site activation (without GTP no polymerization occurs) produces hydrated electrons (they are detected by the UV spectra), which are trapped in the shell of biological water, surrounding the tubulin. These electrons generate an additional, nonlinear by nature, polarization effect, responsible for the second harmonic generation at lambda=365 nm (the first harmonic is centered at lambda=730 nm) and manyfold increase in strength of the initial electric field. The results are supported by model calculations, based on the assumption of positive (negative) feedback, appearing on interaction of charge transfer exciton dipoles with the applied electromagnetic field.     

Simulating GTP:Mg and GDP:Mg with a simple force field: A structural and thermodynamic analysis

Di‐ and tri‐phosphate nucleotides are essential cofactors for many proteins, usually in an Mg²⁺‐bound form. Proteins like GTPases often detect the difference between NDP and NTP and respond by changing conformations. To study such complexes, simple, fixed charge force fields have been used, which allow long simulations and precise free energy calculations. The preference for NTP or NDP binding depends on many factors, including ligand structure and Mg²⁺ coordination and the changes they undergo upon binding. Here, we use a simple force field to examine two Mg²⁺ coordination modes for the unbound GDP and GTP: direct, or “Inner Sphere” (IS) coordination by one or more phosphate oxygens and indirect, “Outer Sphere” (OS) coordination involving one or more bridging waters. We compare GTP: and GDP:Mg binding with OS and IS coordination; combining the results with experimental data then indicates that GTP prefers the latter. We also examine different kinds of IS coordination and their sensitivity to a key force field parameter: the optimal Mg:oxygen van der Waals distance R_min. Increasing R_min improves the Mg:oxygen distances, the GTP: and GDP:Mg binding affinities, and the fraction of GTP:Mg with β + γ phosphate coordination, but does not improve or change the GTP/GDP affinity difference, which remains much larger than experiment. It has no effect on the free energy of GDP binding to a GTPase. © 2012 Wiley Periodicals, Inc.     

A Self‐Assembled Luminescent Host That Selectively Senses ATP in Water

Metal‐ion‐directed self‐assembly has been used to construct kinetically inert, water‐soluble heterometallic Ru₂Re₂ hosts that are potential sensors for bioanions. A previously reported metallomacrocycle and a new derivative synthesised by this approach are found to be general sensors for bioanions in water, showing an “off–on” luminescent change that is selective for nucleotides over uncharged nucleobases. Through a change in the ancillary ligands coordinated to the ruthenium centres of the host, an “off–on” sensor has been produced. Whilst this host only shows a modest enhancement in binding affinities for nucleotides relative to the other two host systems, its sensing response is much more specific. Although a distinctive “off–on” luminescence response is observed for the addition of adenosine triphosphosphate (ATP), related structures such as adenine and guanosine triphosphate (GTP) do not induce any emission change in the host. Detailed and demanding DFT studies on the ATP‐ and GTP‐bound host–guest complexes reveal subtle differences in their geometries that modulate the stacking interactions between the nucleotide guests and the ancillary ligands of the host. It is suggested that this change in stacking geometries affects solvent accessibility to the binding pocket of the host and thus leads to observed difference in the host luminescence response to the guests.     

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non‐radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine‐, vinblastine‐ and paclitaxel‐binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin‐binding site of 4‐substituted methoxylbenzoyl‐aryl‐thiazoles (SMART‐H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART‐H specifically and reversibly bound only to the colchicine‐binding site, but not to vinblastine‐ or paclitaxel sites. This new non‐radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in β-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αβα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and β-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αβ-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.     

A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.     

Excitation of vibrations in microtubules in living cells

Microtubules, which are thought to be the primary organizers of the cytoskeleton, are electrical polar structures with extraordinary elastic deformability at low stress and with energy supply from hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). At least a part of the energy supplied from hydrolysis can excite vibrations. Energy is mainly lost by viscous damping of the surrounding cytosol. Viscous damping is diminished by a slip layer which is formed by an attracted ionic charge layer and by a thin surface layer of the microtubule. Relaxation time caused by viscous damping may be several orders of magnitude greater than period of vibrations at 10 MHz. Energy supplied to the microtubule is of the order of magnitude of 10(-14) W cm(-1) (per unit length of the microtubule).     

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Aptamers selected for higher-affinity binding are not more specific for the target ligand

Previous study of eleven different in vitro-selected RNA aptamers that bind guanosine triphosphate (GTP) with K(d)s ranging from 8 microM to 9 nM showed that more information is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural solutions to a functional problem that are are as simple as possible in terms of the information content needed to define them. It has long been assumed that the simplest way to improve the affinity of an aptamer is to increase the shape and functional group complementarity of the RNA binding pocket for the ligand. This argument underlies the hypothesis that selection for higher-affinity aptamers automatically leads to structures that bind more specifically to the target molecule. Here, we examined the binding specificities of the eleven GTP aptamers by carrying out competition binding studies with sixteen different chemical analogues of GTP. The aptamers have distinct patterns of specificity, implying that each RNA is a structurally unique solution to the problem of GTP binding. However, these experiments failed to provide evidence that higher-affinity aptamers bind more specifically to GTP. We suggest that the simplest way to improve aptamer K(d)s may be to increase the stability of the RNA tertiary structure with additional intramolecular RNA-RNA interactions; increasingly specific ligand binding may emerge only in response to direct selection for specificity.     

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Molecular Recognition of Nucleotides in Water by Scorpiand‐Type Receptors Based on Nucleobase Discrimination

The detection of nucleotides is of crucial importance because they are the basic building blocks of nucleic acids. Scorpiand‐based polyamine receptors functionalized with pyridine or anthracene units are able to form stable complexes with nucleotides in water, based on coulombic, π–π stacking, and hydrogen‐bonding interactions. This behavior has been rationalized by means of an exploration with NMR spectroscopy and DFT calculations. Binding constants were determined by potentiometry. Fluorescence spectroscopy studies have revealed the potential of these receptors as sensors to effectively and selectively distinguish guanosine‐5′‐triphosphate (GTP) from adenosine‐5′‐triphosphate (ATP).     

Recognizing five molecular ligand-binding sites with similar chemical structure

Accurate identification of ligand-binding sites and discovering the protein–ligand interaction mechanism are important for understanding proteins' functions and designing new drugs. Meanwhile, accurate computational prediction and mechanism research are two grand challenges in proteomics. In this article, ligand-binding residues of five ligands (ATP, ADP, GTP, GDP, and NAD) are predicted as a group, due to their similar chemical structures and close biological function relations. The data set of binding sites by five ligands (ATP, ADP, GTP, GDP, and NAD) are collated from Biolip database. Then, five features, containing increment of diversity value, matrix scoring value, auto-covariance, secondary structure information, and surface accessibility information are used in binding site predictions. The support vector machine (SVM) model is used with the five features to predict ligand-binding sites. Finally, prediction results are tested by fivefold cross validation. Accuracy (Acc) of five ligands (ATP, ADP, GTP, GDP, and NAD) achieves 77.4%, 71.2%, 82.1%, 82.9%, and 85.3%, respectively; and Matthew correlation coefficient (MCC) of the above five ligands achieves 0.549, 0.424, 0.643, 0.659, and 0.702, respectively. The research result shows that for ligands with similar chemical structures, microenvironment of their binding sites and their sensitivities to features are similar, while, differences of their ligand-binding properties exist at the same time. © 2019 Wiley Periodicals, Inc.     

Impacts of delignification and hot water pretreatment on the water induced cell wall swelling behavior of grasses and its relation to cellulolytic enzyme hydrolysis and binding

The relationships between biomass composition, water retention value (WRV), settling volume and enzymatic glucose yield and enzyme binding is investigated in this work by employing grasses pretreated with combinations of alkaline hydrogen peroxide (AHP) delignification and liquid hot water pretreatment that result in significant alterations of cell wall properties and subsequent enzymatic hydrolysis yields. Specifically, these cell wall treatments are performed on corn stover and switchgrass to generate material with a range of lignin (6–35 %) and xylan (2–28 %) contents as well as a range of other properties such as carboxylic acid content, water binding affinity and swellability. It was determined that WRV and settling volume are predictors of glucose yield (R² = 0.900 and 0.895 respectively) over the range of materials and treatment conditions used. It was also observed that mild AHP delignification can result in threefold increases in the WRV. Dynamic vapor sorption isotherms demonstrated that AHP-delignified corn stover exhibited an increased affinity for water sorption from the vapor phase relative to untreated corn stover. These results indicate that these water properties may be useful proxies for biomass susceptibility to enzymatic deconstruction.     

A theoretical study on the structure and hygroscopicity of ammonium dinitramide

Density functional theory (DFT) and the dispersion corrected DFT have been used to investigate the hygroscopicity of ammonium dinitramide (ADN). Calculation results show that the gaseous ADN has a strong hydrogen bond. But the ionic pair structure NH₄ ⁺ · N(NO₂)^? is stabilized upon the addition of water molecules. Natural bond orbital calculations suggest that the intra- and intermolecular orbital interactions LP(O) → σ*(N–H) or LP(O) → σ*(O–H) make the system stabilized as a whole. En energy decomposition analysis reveals that the interactions between ADN and H₂O are dominated by the electrostatic and orbital interactions. The formation reactions become more spontaneous with the increasing number of water molecules but can be weakened by the growing temperature from 200 to 400 K. Moreover, the molecular dynamic method is applied to explore a more realistic cluster model to study the interactions between ADN and H₂O.     

Relationship between orbital energy gaps and excitation energies for long‐chain systems

The difference between the excitation energies and corresponding orbital energy gaps, the exciton binding energy, is investigated based on time‐dependent (TD) density functional theory (DFT) for long‐chain systems: all‐trans polyacetylenes and linear oligoacenes. The optimized geometries of these systems indicate that bond length alternations significantly depend on long‐range exchange interactions. In TDDFT formalism, the exciton binding energy comes from the two‐electron interactions between occupied and unoccupied orbitals through the Coulomb‐exchange‐correlation integral kernels. TDDFT calculations show that the exciton binding energy is significant when long‐range exchange interactions are involved. Spin‐flip (SF) TDDFT calculations are then carried out to clarify double‐excitation effects in these excitation energies. The calculated SF‐TDDFT results indicate that double‐excitation effects significantly contribute to the excitations of long‐chain systems. The discrepancies between the vertical ionization potential minus electron affinity (IP–EA) values and the HOMO–LUMO excitation energies are also evaluated for the infinitely long polyacetylene and oligoacene using the least‐square fits to estimate the exciton binding energy of infinitely long systems. It is found that long‐range exchange interactions are required to give the exciton binding energy of the infinitely long systems. Consequently, it is concluded that long‐range exchange interactions neglected in many DFT calculations play a crucial role in the exciton binding energies of long‐chain systems, while double‐excitation correlation effects are also significant to hold the energy balance of the excitations. © 2016 Wiley Periodicals, Inc.     

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of ?6.4, ?7.2, ?7.4 and ?7.3 kcal/mol with αβ_I, αβ_II, αβ_III and αβ_IV isotypes, respectively. Similarly 10 showed higher binding affinity of ?6.8 kcal/mol with αβ_V, whereas 8 had the highest binding affinity of ?7.2, ?7.1 and ?7.2 kcal/mol, respectively with αβ_VI, αβ_VII and αβ_VIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of ?46.2 and ?38.1 kcal/mol against αβ_III (overexpression of αβ_III has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.     

Dielectric effect on thermodynamic properties in vinblastine by DFT/Onsager modelling

We know that vinblastine, as one of the vinca alkaloids, has played a major role in cancer chemotherapy by inhibiting the polymerisation of tubulin into microtubules. We present an in-depth investigation of the structural, infrared (IR) spectra of vinblastine. The structure of vinblastine is theoretically investigated using the density functional theory and Hartree–Fock levels of theory with the standard 6-31g* and 6-31g** basis sets. The vibrational spectral data obtained from IR spectra are assigned modes based on the results of the theoretical calculations as intensity and frequency curves. The fundamental vibrational modes were characterised depending on the stability of vinblastine in different dielectric constants. Thermodynamic analysis of data demonstrates good correlation of vinblastine at various media by SCRF–Onsager model with linear coefficient (R ²). Thus, the goal of this article is to evaluate and quantify the molecular basis for vinblastine structure in variant positions.     

Purification of guanosine triphosphate cyclohydrolase I from Escherichia coli. The use of competitive inhibitors versus substrate as ligands in affinity chromatography

Different affinity chromatography ligands have been compared for the purification of guanosine triphosphate (GTP) cyclohydrolase I, an enzyme that catalyses the transformation of GTP into formate and dihydroneopterin triphosphate, the first metabolite in the biosynthetic pathway of the pterins. When this enzyme is purified by affinity chromatography on GTP-Sepharose a major fraction of the activity is lost and the yield of enzyme decreases as the amount of enzyme applied to the column decreases. The use of nucleotide competitive inhibitors (UTP and ATP) as ligands in the affinity column has shown that the extent of inactivation of the enzyme is related to the affinity of the enzyme for the ligand. Further, the extent of inactivation was reduced by reducing the length of the columns when using the same volume of GTP-Sepharose. Dihydrofolate-Sepharose gave consistently higher yields of GTP cyclohydrolase I regardless of the amount of enzyme applied, but several other proteins were also obtained. For a high purification of GTP cyclohydrolase I the best yield may be obtained with UTP as the affinity ligand and with the shortest length possible of the affinity column, and the purity of enzyme is comparable with that obtained with GTP-Sepharose.