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RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralog's distinct splicing activities. To this end, we conducted bioinformatics analysis to determine the evolutionary conservation of RRMs versus linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

Hydration free energies of small molecules are commonly used as benchmarks for solvation models. However, errors in predicting hydration free energies are partially due to the force fields used and not just the solvation model. To address this, we have used the 3D reference interaction site model (3D-RISM) of molecular solvation and existing benchmark explicit solvent calculations with a simple element count correction (ECC) to identify problems with the non-bond parameters in the general AMBER force field (GAFF). 3D-RISM was used to calculate hydration free energies of all 642 molecules in the FreeSolv database, and a partial molar volume correction (PMVC), ECC, and their combination (PMVECC) were applied to the results. The PMVECC produced a mean unsigned error of 1.01±0.04kcal/mol and root mean squared error of 1.44±0.07kcal/mol, better than the benchmark explicit solvent calculations from FreeSolv, and required less than 15 s of computing time per molecule on a single CPU core. Importantly, parameters for PMVECC showed systematic errors for molecules containing Cl, Br, I, and P. Applying ECC to the explicit solvent hydration free energies found the same systematic errors. The results strongly suggest that some small adjustments to the Lennard-Jones parameters for GAFF will lead to improved hydration free energy calculations for all solvent models.

The ryanodine receptor type 2 (RyR2) is composed of four subunits that control calcium (Ca) release in cardiac cells. RyR2 serves primarily as a Ca sensor and can respond to rapid sub-millisecond pulses of Ca while remaining shut at resting concentrations. However, it is not known how the four subunits interact for the RyR2 to function as an effective Ca sensor. To address this question, and to understand the role of subunit cooperativity in Ca-mediated signal transduction, we have developed a computational model of the RyR2 composed of four interacting subunits. We first analyze the statistical properties of a single RyR2 tetramer, where each subunit can exist in a closed or open conformation. Our findings indicate that the number of subunits in the open state is a crucial parameter that dictates RyR2 kinetics. We find that three or four open subunits are required for the RyR2 to harness cooperative interactions to respond to sub-millisecond changes in Ca, while at the same time remaining shut at the resting Ca levels in the cardiac cell. If the required number of open subunits is lowered to one or two, the RyR2 cannot serve as a robust Ca sensor, as the large cooperativity required to stabilize the closed state prevents channel activation. Using this four-subunit model, we analyze the kinetics of Ca release from a RyR2 cluster. We show that the closure of a cluster of RyR2 channels is highly sensitive to the balance of cooperative interactions between closed and open subunits. Based on this result, we analyze how specific interactions between RyR2 subunits can induce persistent Ca leak from the sarcoplasmic reticulum (SR), which is believed to be arrhythmogenic. Thus, these results provide a framework to analyze how a pharmacologic or genetic modification of RyR2 subunit cooperativity can induce abnormal Ca cycling that can potentially lead to life-threatening arrhythmias., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mutations in the cardiac ryanodine receptor type 2 (RyR2) have been linked to fatal cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). While many CPVT mutations are associated with an increase in Ca 2+ leak from the sarcoplasmic reticulum, the mechanistic details of RyR2 channel gating are not well understood, and this poses a barrier in the development of new pharmacological treatments. To address this, we explore the gating mechanism of the RyR2 using molecular dynamics (MD) simulations. We test the effect of changing the conformation of certain structural elements by constructing chimera RyR2 structures that are derived from the currently available closed and open cryo-electron microscopy (cryo-EM) structures, and we then use MD simulations to relax the system. Our key finding is that the position of the S4-S5 linker (S4S5L) on a single subunit can determine whether the channel as a whole is open or closed. Our analysis reveals that the position of the S4S5L is regulated by interactions with the U-motif on the same subunit and with the S6 helix on an adjacent subunit. We find that, in general, channel gating is crucially dependent on high percent occupancy interactions between adjacent subunits. We compare our interaction analysis to 49 CPVT1 mutations in the literature and find that 73% appear near a high percent occupancy interaction between adjacent subunits. This suggests that disruption of cooperative, high percent occupancy interactions between adjacent subunits is a primary cause of channel leak and CPVT in mutant RyR2 channels.

The 3D reference interaction site model (3D-RISM) of molecular solvation is a powerful tool for computing the equilibrium thermodynamics and density distributions of solvents, such as water and co-ions, around solute molecules. However, 3D-RISM solutions can be expensive to calculate, especially for proteins and other large molecules where calculating the potential energy between solute and solvent requires more than half the computation time. To address this problem, we have developed and implemented treecode summation for long-range interactions and analytically corrected cut-offs for short-range interactions to accelerate the potential energy and long-range asymptotics calculations in non-periodic 3D-RISM in the AmberTools molecular modeling suite. For the largest single protein considered in this work, tubulin, the total computation time was reduced by a factor of 4. In addition, parallel calculations with these new methods scale almost linearly and the iterative solver remains the largest impediment to parallel scaling. To demonstrate the utility of our approach for large systems, we used 3D-RISM to calculate the solvation thermodynamics and density distribution of 7-ring microtubule, consisting of 910 tubulin dimers, over 1.2 million atoms., (© 2022 Wiley Periodicals LLC.)

The solvent can occupy up to ∼70% of macromolecular crystals, and hence, having models that predict solvent distributions in periodic systems could improve the interpretation of crystallographic data. Yet, there are few implicit solvent models applicable to periodic solutes, and crystallographic structures are commonly solved assuming a flat solvent model. Here, we present a newly developed periodic version of the 3D-reference interaction site model (RISM) integral equation method that is able to solve efficiently and describe accurately water and ion distributions in periodic systems; the code can compute accurate gradients that can be used in minimizations or molecular dynamics simulations. The new method includes an extension of the Ornstein-Zernike equation needed to yield charge neutrality for charged solutes, which requires an additional contribution to the excess chemical potential that has not been previously identified; this is an important consideration for nucleic acids or any other charged system where most or all the counter- and co-ions are part of the "disordered" solvent. We present several calculations of proteins, RNAs, and small molecule crystals to show that x-ray scattering intensities and the solvent structure predicted by the periodic 3D-RISM solvent model are in closer agreement with the experiment than are intensities computed using the default flat solvent model in the refmac5 or phenix refinement programs, with the greatest improvement in the 2 to 4 Å range. Prospects for incorporating integral equation models into crystallographic refinement are discussed.

Mutations in the cardiac ryanodine receptor type 2 (RyR2) have been linked to a variety of cardiac arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). RyR2 is regulated by calmodulin (CaM), and mutations that disrupt their interaction can cause aberrant calcium release, leading to an arrhythmia. It was recently shown that increasing the RyR2-CaM binding affinity could rescue a defective CPVT-related RyR2 channel to near wild-type behavior. However, the interactions that determine the binding affinity at the RyR2-CaM binding interface are not well understood. In this study, we identify the key domains and interactions, including several new interactions, involved in the binding of CaM to RyR2. Also, our comparison between the wild-type and V3599K mutant suggests how the RyR2-CaM binding affinity can be increased via a change in the central and N-terminal lobe binding contacts for CaM. This computational approach provides new insights into the effect of a mutation at the RyR2-CaM binding interface, and it may find utility in drug design for the future treatment of cardiac arrhythmias.

SARS-CoV-2 recently jumped species and rapidly spread via human-to-human transmission to cause a global outbreak of COVID-19. The lack of effective vaccine combined with the severity of the disease necessitates attempts to develop small molecule drugs to combat the virus. COVID19_GIST_HSA is a freely available online repository to provide solvation thermodynamic maps of COVID-19-related protein small molecule drug targets. Grid inhomogeneous solvation theory maps were generated using AmberTools cpptraj-GIST, 3D reference interaction site model maps were created with AmberTools rism3d.snglpnt and hydration site analysis maps were created using SSTMap code. The resultant data can be applied to drug design efforts: scoring solvent displacement for docking, rational lead modification, prioritization of ligand- and protein- based pharmacophore elements, and creation of water-based pharmacophores. Herein, we demonstrate the use of the solvation thermodynamic mapping data. It is hoped that this freely provided data will aid in small molecule drug discovery efforts to defeat SARS-CoV-2.

Electron paramagnetic resonance (EPR) measurements of the rotational diffusion of small nitroxide probes have been demonstrated to be a powerful technique for experimentally investigating the properties of supercooled liquids, such as water. However, since only the rotational diffusion of the probe molecules is measured and EPR measurements are indirect, it is not clear what the relationship between the behavior of water and the probe molecule is. To address this, we have performed molecular dynamics simulations of four nitroxide probes in TIP4P-Ew and OPC water models to directly compare with EPR experiments and to determine the behavior of the water and the underlying microscopic coupling between the water and the probes. In all, 200 ns simulations were run for 23 temperatures between 253 and 283 K for all four probes with each water model for an aggregate of 36.8 μs of simulation time. Simulations for both water models systematically underestimated the rotational diffusion coefficients for both water and probes, though OPC simulations were generally in better agreement with the experiments than TIP4P-Ew simulations. Despite this, when the temperature dependence of the data was fit to a power law, fit parameters for TIP4P-Ew were generally in better agreement with the experiments than OPC. For probe molecules, the singular temperature was found to be T 0 = 226.5 ± 0.4 K from experiments, T 0 = 208 ± 2 K for OPC water, and T 0 = 215 ± 2 K for TIP4P-Ew water. While for water molecules, the singular temperature was found to be T 0 = 220.3 ± 0.2 K from experiments, T 0 = 208 ± 2 K for OPC water, and T 0 = 220 ± 1 K for TIP4P-Ew water. Systematic underestimation of the rotational diffusion coefficients was most pronounced at lower temperatures and was clearly observed in changes to the Arrhenius activation energy. Above the maximum density temperature of T ρ max = 277 K, an activation energy of E A ≈ 16.7 kJ/mol was observed for the probes from experiments, while OPC had E A ≈ 15.2 kJ/mol and TIP4P-Ew had E A ≈ 14.6 kJ/mol. Below the maximum density temperature, the activation energy jumped to E A ≈ 32.5 kJ/mol for experiments but only E A ≈ 23 kJ/mol for OPC and E A ≈ 22 kJ/mol for TIP4P-Ew. In all cases, we saw good agreement between the behavior of the probe molecules and water. To understand why, we calculated the average number of hydrogen bonds between the probe molecules and water. From this, we were able to explain the rotational diffusion times for all of the probes. These results show that current molecular models are sufficient to capture physical phenomena observed with EPR and to help elucidate why the probes provide accurate insights into the behavior of supercooled water.

Computed, high-resolution, spatial distributions of solvation energy and entropy can provide detailed information about the role of water in molecular recognition. While grid inhomogeneous solvation theory (GIST) provides rigorous, detailed thermodynamic information from explicit solvent molecular dynamics simulations, recent developments in the 3D reference interaction site model (3D-RISM) theory allow many of the same quantities to be calculated in a fraction of the time. However, 3D-RISM produces atomic-site, rather than molecular, density distributions, which are difficult to extract physical meaning from. To overcome this difficulty, we introduce a method to reconstruct molecular density distributions from atomic-site density distributions. Furthermore, we assess the quality of the resulting solvation thermodynamics density distributions by analyzing the binding site of coagulation Factor Xa with both GIST and 3D-RISM. We find good qualitative agreement between the methods for oxygen and hydrogen densities as well as direct solute-solvent energetic interactions. However, 3D-RISM predicts lower energetic and entropic penalties for moving water from the bulk to the binding site., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: M.K.G. has an equity interest in and is a cofounder and scientific advisor of VeraChem LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The Ornstein-Zernike (OZ) integral equation theory is a powerful approach to simple liquids due to its low computational cost and the fact that, when combined with an appropriate closure equation, the theory is thermodynamically complete. However, approximate closures proposed to date exhibit pressure or free energy inconsistencies that produce inaccurate or ambiguous results, limiting the usefulness of the Ornstein-Zernike approach. To address this problem, we combine methods to enforce both pressure and free energy consistency to create a new closure approximation and test it for a single-component Lennard-Jones fluid. The closure is a simple power series in the direct and total correlation functions for which we have derived analytical formulas for the excess Helmholtz free energy and chemical potential. These expressions contain a partial molar volumelike term, similar to excess chemical potential correction terms recently developed. Using our bridge approximation, we have calculated the pressure, Helmholtz free energy, and chemical potential for the Lennard-Jones fluid using the Kirkwood charging, thermodynamic integration techniques, and analytic expressions. These results are compared with those from the hypernetted chain equation and the Verlet-modified closure against Monte Carlo and equations-of-state data for reduced densities of ρ^{*}<1 and temperatures of T^{*}=1.5, 2.74, and 5. Our closure shows consistency among all thermodynamic paths, except for one expression of the Gibbs-Duhem relation, whereas the hypernetted chain equation and the Verlet-modified closure exhibit consistency between only a few relations. Accuracy of the closure is comparable to the Verlet-modified closure and a significant improvement to results obtained from the hypernetted chain equation.

Implicit solvent methods for classical molecular modeling are frequently used to provide fast, physics-based hydration free energies of macromolecules. Less commonly considered is the transferability of these methods to other solvents. The Statistical Assessment of Modeling of Proteins and Ligands 5 (SAMPL5) distribution coefficient dataset and the accompanying explicit solvent partition coefficient reference calculations provide a direct test of solvent model transferability. Here we use the 3D reference interaction site model (3D-RISM) statistical-mechanical solvation theory, with a well tested water model and a new united atom cyclohexane model, to calculate partition coefficients for the SAMPL5 dataset. The cyclohexane model performed well in training and testing ([Formula: see text] for amino acid neutral side chain analogues) but only if a parameterized solvation free energy correction was used. In contrast, the same protocol, using single solute conformations, performed poorly on the SAMPL5 dataset, obtaining [Formula: see text] compared to the reference partition coefficients, likely due to the much larger solute sizes. Including solute conformational sampling through molecular dynamics coupled with 3D-RISM (MD/3D-RISM) improved agreement with the reference calculation to [Formula: see text]. Since our initial calculations only considered partition coefficients and not distribution coefficients, solute sampling provided little benefit comparing against experiment, where ionized and tautomer states are more important. Applying a simple [Formula: see text] correction improved agreement with experiment from [Formula: see text] to [Formula: see text], despite a small number of outliers. Better agreement is possible by accounting for tautomers and improving the ionization correction.

The composition of the ion atmosphere surrounding nucleic acids affects their folding, condensation and binding to other molecules. It is thus of fundamental importance to gain predictive insight into the formation of the ion atmosphere and thermodynamic consequences when varying ionic conditions. An early step toward this goal is to benchmark computational models against quantitative experimental measurements. Herein, we test the ability of the three dimensional reference interaction site model (3D-RISM) to reproduce preferential interaction parameters determined from ion counting (IC) experiments for mixed alkali chlorides and dsDNA. Calculations agree well with experiment with slight deviations for salt concentrations >200 mM and capture the observed trend where the extent of cation accumulation around the DNA varies inversely with its ionic size. Ion distributions indicate that the smaller, more competitive cations accumulate to a greater extent near the phosphoryl groups, penetrating deeper into the grooves. In accord with experiment, calculated IC profiles do not vary with sequence, although the predicted ion distributions in the grooves are sequence and ion size dependent. Calculations on other nucleic acid conformations predict that the variation in linear charge density has a minor effect on the extent of cation competition., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ionic atmosphere around nucleic acids remains only partially understood at atomic-level detail. Ion counting (IC) experiments provide a quantitative measure of the ionic atmosphere around nucleic acids and, as such, are a natural route for testing quantitative theoretical approaches. In this article, we replicate IC experiments involving duplex DNA in NaCl(aq) using molecular dynamics (MD) simulation, the three-dimensional reference interaction site model (3D-RISM), and nonlinear Poisson-Boltzmann (NLPB) calculations and test against recent buffer-equilibration atomic emission spectroscopy measurements. Further, we outline the statistical mechanical basis for interpreting IC experiments and clarify the use of specific concentration scales. Near physiological concentrations, MD simulation and 3D-RISM estimates are close to experimental results, but at higher concentrations (>0.7 M), both methods underestimate the number of condensed cations and overestimate the number of excluded anions. The effect of DNA charge on ion and water atmosphere extends 20-25 Å from its surface, yielding layered density profiles. Overall, ion distributions from 3D-RISMs are relatively close to those from corresponding MD simulations, but with less Na(+) binding in grooves and tighter binding to phosphates. NLPB calculations, on the other hand, systematically underestimate the number of condensed cations at almost all concentrations and yield nearly structureless ion distributions that are qualitatively distinct from those generated by both MD simulation and 3D-RISM. These results suggest that MD simulation and 3D-RISM may be further developed to provide quantitative insight into the characterization of the ion atmosphere around nucleic acids and their effect on structure and stability., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Using the dielectrically consistent reference interaction site model (DRISM) of molecular solvation, we have calculated structural and thermodynamic information of alkali-halide salts in aqueous solution, as a function of salt concentration. The impact of varying the closure relation used with DRISM is investigated using the partial series expansion of order-n (PSE-n) family of closures, which includes the commonly used hypernetted-chain equation (HNC) and Kovalenko-Hirata closures. Results are compared to explicit molecular dynamics (MD) simulations, using the same force fields, and to experiment. The mean activity coefficients of ions predicted by DRISM agree well with experimental values at concentrations below 0.5 m, especially when using the HNC closure. As individual ion activities (and the corresponding solvation free energies) are not known from experiment, only DRISM and MD results are directly compared and found to have reasonably good agreement. The activity of water directly estimated from DRISM is nearly consistent with values derived from the DRISM ion activities and the Gibbs-Duhem equation, but the changes in the computed pressure as a function of salt concentration dominate these comparisons. Good agreement with experiment is obtained if these pressure changes are ignored. Radial distribution functions of NaCl solution at three concentrations were compared between DRISM and MD simulations. DRISM shows comparable water distribution around the cation, but water structures around the anion deviate from the MD results; this may also be related to the high pressure of the system. Despite some problems, DRISM-PSE-n is an effective tool for investigating thermodynamic properties of simple electrolytes.

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis., (Copyright © 2011 Wiley-Liss, Inc.)

We have modified the popular MM/PBSA or MM/GBSA approaches (molecular mechanics for a biomolecule, combined with a Poisson-Boltzmann or generalized Born electrostatic and surface area nonelectrostatic solvation energy) by employing instead the statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D reference interaction site model, or 3D-RISM-KH) coupled with molecular mechanics or molecular dynamics ( Blinov , N. ; et al. Biophys. J. 2010 ; Luchko , T. ; et al. J. Chem. Theory Comput. 2010 ). Unlike the PBSA or GBSA semiempirical approaches, the 3D-RISM-KH theory yields a full molecular picture of the solvation structure and thermodynamics from the first principles, with proper account of chemical specificities of both solvent and biomolecules, such as hydrogen bonding, hydrophobic interactions, salt bridges, etc. We test the method on the binding of seven biotin analogues to avidin in aqueous solution and show it to work well in predicting the ligand-binding affinities. We have compared the results of 3D-RISM-KH with four different generalized Born and two Poisson-Boltzmann methods. They give absolute binding energies that differ by up to 208 kJ/mol and mean absolute deviations in the relative affinities of 10-43 kJ/mol.

We present the three-dimensional molecular theory of solvation (also known as 3D-RISM) coupled with molecular dynamics (MD) simulation by contracting solvent degrees of freedom, accelerated by extrapolating solvent-induced forces and applying them in large multi-time steps (up to 20 fs) to enable simulation of large biomolecules. The method has been implemented in the Amber molecular modeling package, and is illustrated here on alanine dipeptide and protein G.

Combination chemotherapy involving Cisplatin is a standard treatment for many cancers. However, following an initial positive response, patients will often relapse, presenting with Cisplatin-resistant disease. One possible mechanism for the acquired resistance to Cisplatin is an increase in DNA repair through the up-regulation of ERCC1, an essential component of the nucleotide excision repair complex. Recruitment of ERCC1 to the site of DNA damage is coordinated through its interaction with a protein known as XPA. As there are currently no effective inhibitors of this interaction, inhibition of the ERCC1/XPA interaction may provide an effective strategy for overcoming the development of Cisplatin-resistant cancers. To discover small molecule inhibitors of this interaction, we have screened both the NCI diversity set of ligands and DrugBank-small molecules against the XPA binding site in ERCC1. These compounds were screened using two different techniques in AUTODOCK to account for receptor flexibility. First, using a set of flexible residues, as determined from MD simulations of the XPA/ERCC1 complex and second, using the relaxed complex scheme implemented by performing independent docking experiments against an ensemble of target conformations that were generated from MD simulations. Lowest energy poses from the two different methods were then used to construct a pharmacophore model, which was then validated by comparison to UCN-01, a weak inhibitor of ERCC1 mediated nucleotide excision.

The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of alphabeta-tubulin. While this has been appreciated for many years, there is ongoing debate over the molecular basis of this instability and the possible role of altered nucleotide occupancy in the induction of a conformational change in tubulin. The debate has organized around seemingly contradictory models. The allosteric model invokes nucleotide-dependent states of curvature in the free tubulin dimer, such that hydrolysis leads to pronounced bending and thus disruption of the lattice. The more recent lattice model describes a predominant role for the lattice in straightening free dimers that are curved regardless of their nucleotide state. In this model, lattice-bound GTP-tubulin provides the necessary force to straighten an incoming dimer. Interestingly, there is evidence for both models. The enduring nature of this debate stems from a lack of high-resolution data on the free dimer. In this study, we have prepared alphabeta-tubulin samples at high dilution and characterized the nature of nucleotide-induced conformational stability using bottom-up hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation experiments. These experiments were accompanied by molecular dynamics simulations of the free dimer. We demonstrate an intermediate state unique to GDP-tubulin, suggestive of the curved colchicine-stabilized structure at the intradimer interface but show that intradimer flexibility is an important property of the free dimer regardless of nucleotide occupancy. Our results indicate that the assembly properties of the free dimer may be better described on the basis of this flexibility. A blended model of assembly emerges in which free-dimer allosteric effects retain importance, in an assembly process dominated by lattice-induced effects.

Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.

Several isotypes of the structural protein tubulin have been characterized. Their expression offers a plausible explanation for differences regarding microtubule function. Although sequence variation between tubulin isotypes occurs throughout the entire protein, it is the extreme carboxy-terminal tails (CTTs) that exhibit the greatest concentration of differences. In humans, the CTTs range in length from 9 to 25 residues and because of a considerable number of glutamic acid residues, contain over 1/3 of tubulin's total electrostatic charge. The CTTs are believed to be highly disordered and their precise function has yet to be determined. However, their absence has been shown to result in altered microtubule stability and a reduction in the interaction with several microtubule-associated proteins (MAPs). To characterize the role that CTTs play in microtubule function, we examined the global conformational differences within a set of nine human beta-tubulin isotypes using replica exchange molecular dynamics simulations. Through the analysis of the resulting configuration ensembles, we quantified differences such as the CTTs sequence influence on overall flexibility and average secondary structure. Although only minor variations between each CTT were observed, we suggest that these differences may be significant enough to affect interactions with MAPs, thereby influencing important properties such as microtubule assembly and stability.

Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.

This paper discusses the results of homology modeling and resulting calculation of key structural and physical properties for close to 300 tubulin sequences, including alpha, beta, gamma, delta and epsilon -tubulins. The basis for our calculations was the structure of the tubulin dimer published several years ago by Nogales et al. (1998), later refined to 3.5 resolution by Lowe et al. (2001). While, it appears that the alpha, beta and gamma-tubulins segregate into distinct structural families, we have found several differences in the physical properties within each group. Each of the alpha, beta and gamma- tubulin groups exhibit major differences in their net electric charge, dipole moments and dipole vector orientations. These properties could influence functional characteristics such as microtubule stability and assembly kinetics, due to their effects on the strength of protein-protein interactions. In addition to the general structural trends between tubulin isoforms, we have observed that the carboxy-termini of alpha and beta-tubulin exists in at least two stable configurations, either projecting away from the tubulin (or microtubule) surface, or collapsed onto the surface. In the latter case, the carboxy-termini form a lattice distinctly different from that of the well-known A and B lattices formed by the tubulin subunits. However, this C-terminal lattice is indistinguishable from the lattice formed when the microtubule-associated protein tau binds to the microtubule surface. Finally, we have discussed how tubulin sequence diversity arose in evolution giving rise to its particular phylogeny and how it may be used in cell- and tissue-specific expression including embryonal development.

The levels of phosphate esters and the activities of thiamine biotransformation enzymes in the blood and tissues of albino rats were studied during oxythiamine-induced B1 deficiency and after metabolic correction with thiamine and taurine. Among thiamine phosphates, the most informative indicators of thiamine deficiency were shown to be triphosphate esters and free thiamine diphosphate. The biosynthetic enzymes thiamine kinase and thiamine diphosphate kinase played a decisive role in maintaining the initial rate and in recovering the physiologically active forms of vitamin B1. The activation of hydrolytic enzymes of thiamine phosphate esters occurred by producing abundant free thiamine diphosphate and thiamine triphosphate. Within the first hours, taurine favoured the acceleration of phosphoester biosynthesis and, accumulating in the tissues, inhibited vitamin phosphorylation reactions.