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The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPAR[gamma]-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin-and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass., Introduction Skeletal loading and unloading have major effects on skeletal homeostasis. In contrast to postmenopausal osteoporosis, in which bone resorption plays a major role in bone loss, and age-related osteoporosis [...]

The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype, nhaA over-expression mostly confers enhanced sodium ([Na.sup.+]) tolerance and not acetate ([Ac.sup.-]) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated [Na.sup.+] and [Ac.sup.-] ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies. ethanol | inhibitor | microarray | sequencing | systems biology doi/ 10.1073/pnas.0914506107

Microscopic statistical pressure fluctuations can, in principle, lead to corresponding fluctuations in the shape of a protein energy landscape. To examine this, nanosecond molecular dynamics simulations of lysozyme are performed covering a range of temperatures and pressures. The well known dynamical transition with temperature is found to be pressure-independent, indicating that the effective energy barriers separating conformational substates are not significantly influenced by pressure. In contrast, vibrations within substates stiffen with pressure, due to increased curvature of the local harmonic potential in which the atoms vibrate. The application of pressure is also shown to selectively increase the damping of the anharmonic, low-frequency collective modes in the protein, leaving the more local modes relatively unaffected. The critical damping frequency, i.e., the frequency at which energy is most efficiently dissipated, increases linearly with pressure. The results suggest that an invariant description of protein energy landscapes should be subsumed by a fluctuating picture and that this may have repercussions in, for example, mechanisms of energy dissipation accompanying functional, structural, and chemical relaxation. critical damping | dynamical transition | energy dissipation | harmonic-anharmonic motions

Recent work has shown that the nature of hydration of pure hydrophobic surfaces changes with the length scale considered: water hydrogen-bonding networks adapt to small exposed hydrophobic species, hydrating or 'wetting' them at relatively high densities, whereas larger hydrophobic areas are 'dewetted' [Chandler D (2005), Nature 29:640-647]. Here we determine whether this effect is also present in peptides by examining the folding of a [beta]-hairpin (the 14-residue amyloidogenic prion protein H1 peptide), using microsecond time-scale molecular dynamics simulations. Two simulation models are compared, one explicitly including the water molecules, which may thus adapt locally to peptide configurations, and the other using a popular continuum approximation, the generalized Born/surface area implicit solvent model. The results obtained show that, in explicit solvent, peptide conformers with high solvent-accessible hydrophobic surface area indeed also have low hydration density around hydrophobic residues, whereas a concomitant higher hydration density around hydrophilic residues is observed. This dewetting effect stabilizes the fully folded [beta]-hairpin state found experimentally. In contrast, the implicit solvent model destabilizes the fully folded hairpin, tending to cluster hydrophobic residues regardless of the size of the exposed hydrophobic surface. Furthermore, the rate of the conformational transitions in the implicit solvent simulation is almost doubled with respect to that of the explicit solvent. The results suggest that dehydration-driven solvent exposure of hydrophobic surfaces may be a significant factor determining peptide conformational equilibria. hydrophobicity | hydration density | molecular dynamics simulation | explicit and implicit solvent model | [beta]-hairpin

Molecular dynamics simulation study is used to investigate the differential effects of three closely-related sterols, cholesterol, ergosterol and lanosterol on the dipalmitoyl phosphatidylcholine membrane. The findings prove that ergosterol is the most efficient of all three, as it helps in promoting the liquid-ordered phase, as well as the lipid domain formation in the membrane.

The catalysis mechanism in myosin II is investigated by using quantum mechanical/molecular mechanical reaction path calculations. The result has revealed a key element of the chemomehcanical coupling that underlies the motor cycle, such as the modulation of actin unbinding or binding in response to the ATP or ADP.P1 state of nucleotide-bound myosin.

Molecular dynamics (MD) simulations and normal-mode analyses are performed on various models of crystalline myoglobin in order to characterize intra- and inter-protein vibrations at 150 K. An accurate description of the environment is found to be essential for reproducing the experimental Boson peak form and position.

The chi(C(sub alpha)-C(sub beta) torsional barrier in the dipeptide alanine (N-methyl-L-alanyl-N-methylamide) crystal was investigated using ab initio calculations at various levels of theory, molecular mechanics, and molecular dynamics. It is suggested that environment-catalyzed rotation of methyl groups can occur both in the solid phase and in native biological structures, though the effect might be temperature-dependent.

A mechanism involving two-metal ions and double Lewis-acid activation of the substrate, combined with leaving group stabilization is proposed, for a number of phosphoryltransfer enzymes, including the exonuclease subunit of DNA polymerase I. It is designed as a synthetic phosphoryl transfer catalyst the dicopper(II)macrocyclic complex LCu2 which is inspired by the active site structure of exonuclease subunit of DNA polymerase I enzyme.

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ([approximately equal to] 60[degrees]) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm. chemo-mechanical coupling | conformational transition | Conjugate Peak Refinement | muscle contraction | power stroke

Characterization of the physical properties of protein surface hydration water is critical for understanding protein structure and folding. Here, using molecular dynamics simulation, we provide an explanation of recent x-ray and neutron solution scattering data that indicate that the density of water on the surface of lysozyme is significantly higher than that of bulk water. The simulation-derived scattering profiles are in excellent agreement with the experiment. In the simulation, the 3-[Angstrom]-thick first hydration layer is 15% denser than bulk water. About two-thirds of this increase is the result of a geometric contribution that would also be present if the water was unperturbed from the bulk. The remaining third arises from modification of the water structure and dynamics, involving approximately equal contributions from shortening of the average water-water O-O distance and an increase in the coordination number. Variation in the first hydration shell density is shown to be determined by topographical and electrostatic properties of the protein surface. On average, denser water is found in depressions on the surface in which the water dipoles tend to be aligned parallel to each other by the electrostatic field generated by the protein atoms.

Conjugate peak refinement analysis of the annexin V shows that Trp187 burial leads to large conformational strain increase and protein-protein interaction energies. Acidic residues are involved in the conformational change causing a succession of hydrogen bonds with TRP187. The pathway involves more than 300 dihedral angle transitions and helix unwinding.

The cyclic peptide antibiotic Gramicidin S is characterized by the sequence cyclo-(Leu-D-Phe-Pro-Val-Orn)sub 2. To shed light on its molecular dynamics, a simulation of Gramicidin S in dimethyl sulfoxide solution is done. Throughout the simulation, the backbone ring is shown to maintain an antiparallel beta-pleated sheet conformation with two-type II beta-turns. The backbone dihedrals are also shown to be relatively inflexible as they do not undergo any conformational transitions.

Infrared and Raman optical spectroscopies are often used to probe the structure and dynamics of biological molecules, however inelastic neturon scattering (INS) is more convenient for testing vibrational and molecular mechanics. Time-focussing crystal analysis was used on the vibrational neutron spectrum of Staphylococcal nuclease (SNase), and the spectrum compared with S(Q,omega) from normal mode analysis. Calculated spectral intensities were in general agreement with experiment and were used to assign the peaks.

The comparison of the structures of toxin alpha and the synthetic octapeptide that produces antibodies against the toxin indicates differences in conformation between the peptide and the toxin. The residues between 23 and 28 and between 37 and 40 form a beta-strand in the toxin while they are not ordered in the peptide. The toxin has a beta-strand between residues 28 and 30 but the peptide has loop structures. The residues between 30 and 33 exhibit a reverse-turn fold in the toxin and the peptide that is probably responsible for the immunogenic activity.

The minimum energy geometries and potential energy changes as a function of conformation are studied in molecules of alkanes and molecules with methyl groups bound to sp2 carbons. Force field parameters are developed for use on macromolecules. Most of the experimental and quantum mechanical values for the geometries and conformational energetics of the molecules can be reproduced via the form of the potential function used. Subtle series effects such as the bond angles in CH3- and CH2- groups in alkanes are also reproduced.

The molecular dynamics (MD) simulations of native and truncated staphylococcal nuclease are compared at 300 K in aqueous solution by utilizing version 23 of the CHARMM program. The truncated form is prepared by removing the terminal residue at 13 C. The three-dimensional structure of the native form remains intact but that of the truncated form undergoes denaturation in a 530 ps MD simulation, thereby resulting to major modifications in the inhibitor binding pocket. The resulting structure is consistent with conformation-dependent chemical cleavage tests.

Molecular dynamics (MD) simulations in both crystalline and aqueous solution environments is utilized to examine the Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme which accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition. The simulation results obtained for the reaction with broad application in organic chemistry demonstrated that full catalytic activity of the reaction depends on the [Mg.sup.2+] ion concentration.

Circular dichroism experiments were performed to understand the lipid bilayer embedded peptide and fully helical state of even at 90 degree [Celsius]. High-temperature simulations of experimentally validated thermostable systems suggested a new pathway for systematic exploration of peptide partitioning properties.

Multiple unbiased, atomistic molecular dynamics (MD) simulations are used for characterizing the folding of a 15-residue [beta]-hairpin peptide (Peptide 1). The peptide is always observed to form partially structured conformations involving a non-native (ESYI) turn from which the native (NPDG) turn has formed, thus triggering the folding to the [beta]-hairpin.

The catalytic mechanism of backbone phosphodiester hydrolysis by the restriction enzyme EcoRV is examined by using hybrid quantum mechanical/molecular mechanical calculations. The protein environment is tuned to electrostatically stabilize the transition state structures and the direct catalytic impact of essential residues is analyzed.

The two major proposed reaction mechanisms of the bacterial organomercurial lyase, MerB, are examined and compared by using hybrid density functional theory calculations. The analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis has shown that MerB has lowered the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.

The changes in the free energy barrier ([DELTA]E), entropy and motional parameters associated with the rotation of methyl groups in a protein (calmodulin (CaM)) on binding a ligand are examined by using molecular dynamics simulation. A correlation is found between rotational barrier changes and proximity to the protein-peptide binding interface and methyl groups displaying large changes in [DELTA]E have reported on elements in the protein undergoing structural change on binding.

Hybrid quantum mechanics/molecular mechanics proton transfer calculations are performed using different protein structures and with different retinal geometries and active site water molecules in order to assess whether the transient protonation of Asp212 has participated in deprotonation of Asp85. The results have shown that a transient [O] conformer with protonated Asp212 is sampled during the long-distance proton transfer to the proton release group.

A set of asparagine mutants in the transmembrane (TM) domain of the erythropoietin receptor (EpoR) is examined by addressing the importance of TM domain packing density for receptor functionality. The results have shown that the packing density of the TM domain has provided a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

Atomistic molecular dynamics simulations are used for examining the mechanisms by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, is bound to ice. The results have shown a dual role for the solvation water around the protein.

A method is developed by using an extended quasi-Gaussian entropy theory parameterized via use of molecular dynamics simulation that provides a complete description of the thermodynamics of peptide conformational states. The method is applied to analyze the conformational thermodynamics of MR121-GSGSW, a peptide that possesses the lowest partial molar entropy.

The atomistic molecular dynamics (MD) simulation is employed to study the significance of methyl groups in the onset of the low-temperature anharmonic dynamics in a crystalline protein. The cavities present in the proteins are found to act as the activation centers of the discussed dynamics.

The internal protein dynamics is described using a model combining Langevin vibration with diffusion. The results have shown that a global description of nanosecond temperature-dependent diffusion and vibrational internal protein dynamics can be obtained by applying the results of the present diffusion-vibration model to the vibrational motions obtained from a normal-mode analysis.

Quantum mechanical/molecular mechanical (QM/MM) reaction-path calculations were used for examining the protein photocycle. The barrier for the thermal cis-trans back-isomerization is lowered by retinal twisting and the rate-limiting barrier for back-isomerization is 5-6 kcal/mol larger than the barrier for the first proton-transfer step.

The modification of internal vibrational modes in a protein due to intraprotein anharmonicity and solvation effects is determined by performing molecular dynamics (MD) simulations of myoglobin. The diagonal and off-diagonal Langevin friction matrix elements, which model the roughness of the vibrational potential energy surfaces, are determined together with the vibrational potentials of mean force from the MD trajectories at 120 K and 300 K in vacuum and in solution.

The Poisson-Boltzman/surface area PB/SA combined with a quantum mechanical description of the ligand in protein/ligand binding affinity calculation is investigated. The result reveals quantum mechanics/molecular mechanics QM/MM is promising new avenue for automated docking and scoring of flexible ligands.

The proton transfer energy profiles in bacteriorhodopsin for various retinal isomeric states are investigated by employing quantum mechanics (QM)/ molecular mechanics (MM) minimum-energy path calculations. The proton transfer energy profiles are determined by two opposite-sign contributions, first the interaction between Asp85 and the 13-cis retinal, strongly favoring proton transfer, and the other arising from the protein electrostatic environment that stabilizes the ion pair state and gives rise to a proton transfer barrier.

The vibrational entropy change on the burial of a crystallographically well-ordered water molecule in bovine pancreatic trypsin inhibitor (BPTI) using normal-mode analysis is dissected. A detailed analysis is performed of how the translational and rotational degrees of freedom of the isolated water molecule are transformed into vibrational modes in the complex.

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in [D.sub.2]O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent-protein systems is much more gradual than in pure [D.sub.2]O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.

A study was conducted to investigate the recognition of a neutron incoherent spin-echo (NISE) signal on a hydrated powder of the enzyme glutamate dehydrogenase. The direct relationship between the scattering function and atomic trajectories was identified as the major biological responsibility of the NISE. The findings affirmed the importance of NISE in the analysis of nanosecond motions in biological specimens, thereby making it a complement of molecular dynamics.