ALBERT

Description: Background: Minor changes in protein structure induced by small organic and inorganic molecules can result in significantmetabolic effects. The effects can be even more profound if the molecular players are chemically active andpresent in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxidedonor (spermine NONOate), ATP and sodium/potassium environment on the dynamics of thermal unfoldingof human hemoglobin (Hb). The effect of these molecules was examined by means of circular dichroismspectrometry (CD) in the temperature range between 25 oC and 70 oC. The alpha-helical content of bufferedhemoglobin samples (0.1 mg/ml) was estimated via ellipticity change measurements at a heating rate of 1oC/min. Results: Major results were: 1) spermine NONOate persistently decreased the hemoglobin unfolding temperature Tuirrespectively of the Na+/K+ environment, 2) ATP instead increased the unfolding temperature by 3 oC inboth sodium-based and potassium-based buffers and 3) mutual effects of ATP and NO were stronglyinfluenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partialunfolding of alpha-helical structures even at room temperature. Conclusion: The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulationof protein activity by small solutes in the cell.

Description: Bacteria dynamically regulate their intricate intracellular organization involving proteins that facilitate cell division, motility, and numerous other processes. Consistent with this sophisticated organization, bacteria are able to create asymmetries and spatial gradients of proteins by localizing signaling pathway components. We use mathematical modeling to investigate the biochemical and physical constraints on the generation of intracellular gradients by the asymmetric localization of a source and a sink. We demonstrate that gradients can be established in a variety of bacterial morphologies such as rods, crescents, spheres, branched and constricted cells. We present a systematic computational analysis of the effects of other regulatory mechanisms, such as synthesis, degradation, saturation, and cell growth. Taken together, these results suggest that gradients are a robust and potentially common mechanism for providing intracellular spatial cues.

Description: Background: Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 mum for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results: Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions: In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519--17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.

Description: Advances in computational biology allow systematic investigations to ascertain whether internal chemical gradients can be maintained in bacteria -- an open question at the resolution limit of fluorescence microscopy. While it was previously believed that the small bacterial cell size and fast diffusion in the cytoplasm effectively remove any such gradient, a new computational study published in BMC Biophysics supports the emerging view that gradients can exist. The study arose from the recent observation that phosphorylated CtrA forms a gradient prior to cell division in Caulobacter crescentus, a bacterium known for its complicated cell cycle. Tropini et al. (2012) postulate that such gradients can provide an internal chemical compass, directing protein localization, cell division and cell development. More specifically, they describe biochemical and physical constraints on the formation of such gradients and explore a number of existing bacterial cell morphologies. These chemical gradients may limit in vitro analyses, and may ensure timing control and robustness to fluctuations during critical stages in cell development.

Description: Background: During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile "trigger loop" of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the "bridge helix" that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results: All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as "switch" residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions: Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

Description: Background: Accurate modeling of electrostatic potential and corresponding energies becomesincreasingly important for understanding properties of biological macromolecules and theircomplexes. However, this is not an easy task due to the irregular shape of biological entitiesand the presence of water and mobile ions. Results: Here we report a comprehensive suite for the well-known Poisson-Boltzmann solver, DelPhi,enriched with additional features to facilitate DelPhi usage. The suite allows for easydownload of both DelPhi executable files and source code along with a makefile for localinstallations. The users can obtain the DelPhi manual and parameter files required for thecorresponding investigation. Non-experienced researchers can download examples containingall necessary data to carry out DelPhi runs on a set of selected examples illustrating variousDelPhi features and demonstrating DelPhi's accuracy against analytical solutions. Conclusions: DelPhi suite offers not only the DelPhi executable and sources files, examples and parameterfiles, but also provides links to third party developed resources either utilizing DelPhi orproviding plugins for DelPhi. In addition, the users and developers are offered a forum toshare ideas, resolve issues, report bugs and seek help with respect to the DelPhi package. Theresource is available free of charge for academic users from URL:http://compbio.clemson.edu/delphi.php

Description: Background: Cell migration plays an essential role in many biological processes, such as cancer metastasis, wound healing and immune response. Cell migration is mediated through protrusion and focal adhesion (FA) assembly, maturation and disassembly. Epidermal growth factor (EGF) is known to enhance migration rates in many cell types; however it is not known how FA maturation, FA dynamics and protrusion dynamics are regulated during EGF-induced migration. Here we use total internal reflection fluorescence (TIRF) microscopy and image analysis to quantify FA properties and protrusion dynamics under different doses of EGF stimulation. Results: EGF was found to broaden the distribution of cell migration rates, generating more fast and slow cells. Furthermore, groups based on EGF stimulation condition or cell migration speed were marked by characteristic signatures. When data was grouped based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was grouped based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells. Conclusions: Only a portion of the FA properties and protrusion dynamics that correlate with migration speed, correlate with the EGF stimulation condition. Those that do not correlate with the EGF stimulation condition constitute the most sensitive output for identifying why these cells respond differently than other cells under the same stimulation conditions. The idea that EGF can both increase and decrease the migration speed of individual cells in a population has particular relevance to cancer metastasis where the microenvironment can select subpopulations based on some adhesion and protrusion characteristics, leading to a more invasive phenotype as would be seen if all cells responded like an "average" cell.

Description: Background: Protein-protein recognition is of fundamental importance in the vast majority of biological processes. However, it has already been demonstrated that it is very hard to distinguish true complexes from false complexes in so-called cross-docking experiments, where binary protein complexes are separated and the isolated proteins are all docked against each other and scored. Does this result, at least in part, reflect a physical reality? False complexes could reflect possible nonspecific or weak associations. Results: In this paper, we investigate the twilight zone of protein-protein interactions, building on an interesting outcome of cross-docking experiments: false complexes seem to favor residues from the true interaction site, suggesting that randomly chosen partners dock in a non-random fashion on protein surfaces. Here, we carry out arbitrary docking of a non-redundant data set of 198 proteins, with more than 300 randomly chosen "probe" proteins. We investigate the tendency of arbitrary partners to aggregate at localized regions of the protein surfaces, the shape and compositional bias of the generated interfaces, and the potential of this property to predict biologically relevant binding sites. We show that the non-random localization of arbitrary partners after protein-protein docking is a generic feature of protein structures. The interfaces generated in this way are not systematically planar or curved, but tend to be closer than average to the center of the proteins. These results can be used to predict biological interfaces with an AUC value up to 0.69 alone, and 0.72 when used in combination with evolutionary information. An appropriate choice of random partners and number of docking models make this method computationally practical. It is also noted that nonspecific interfaces can point to alternate interaction sites in the case of proteins with multiple interfaces. We illustrate the usefulness of arbitrary docking using PEBP (Phosphatidylethanolamine binding protein), a kinase inhibitor with multiple partners. Conclusions: An approach using arbitrary docking, and based solely on physical properties, can successfully identify biologically pertinent protein interfaces.

Description: Background: In the classical view, cell membrane proteins undergo isotropic random motion, that is a 2D Brownian diffusion that should result in an homogeneous distribution of concentration. It is, however, far from the reality: Membrane proteins can assemble into so-called microdomains (sometimes called lipid rafts) which also display a specific lipid composition. Results: The amount of this so-called overconcentration at equilibrium is simply related to the ratio of diffusion coefficients between zones of high and low diffusion. Expanding the model to include particle interaction, we show that inhomogeneous diffusion can impact particles clusterization as well. The clusters of particles were more numerous and appear for a lower value of interaction strength in the zones of low diffusion compared to zones of high diffusion. Conclusion: Provided we assume stable viscosity heterogeneity in the membrane, our model proposes a simple mechanism to explain particle concentration heterogeneity. It has also a non-trivial impact on density of particles when interaction is added. This could potentially have an impact on membrane chemical reactions and oligomerization

Description: Background: The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature. Results: In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount. Conclusions: The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.

Description: Background: Single-particle tracking is a powerful tool for tracking individual particles with high precision. It provides useful information that allows the study of diffusion properties as well as the dynamics of movement. Changes in particle movement behavior, such as transitions between Brownian motion and temporary confinement, can reveal interesting biophysical interactions. Although useful applications exist to determine the paths of individual particles, only a few software implementations are available to analyze these data, and these implementations are generally not user-friendly and do not have a graphical interface,. Results: Here, we present APM_GUI (Analyzing Particle Movement), which is a MatLab-implemented application with a Graphical User Interface. This user-friendly application detects confined movement considering non-random confinement when a particle remains in a region longer than a Brownian diffusant would remain. In addition, APM_GUI exports the results, which allows users to analyze this information using software that they are familiar with. Conclusions: APM_GUI provides an open-source tool that quantifies diffusion coefficients and determines whether trajectories have non-random confinements. It also offers a simple and user-friendly tool that can be used by individuals without programming skills.

Description: Background: The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes. Results: Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution. Conclusions: We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge distribution contributes to the specific short-range interactions responsible for the formation of the bound complex. We also propose a scheme for coreceptor selectivity based on the sequence glycosylation motif, the 11/24/25 rule, and net charge.

Description: Background: The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces. Results: We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated. Conclusions: Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high-affinity interaction with ephrins because the weak interaction with small molecule was found to be insufficient to trigger the transition. Our results therefore highlight the key role of protein dynamics in Eph-ephrin signalling and would benefit future design of agonists/antagonists targeting Eph receptors.

Description: Background: Default activation of the spindle assembly checkpoint provides severe constraints on the underlying biochemical activation rates: on one hand, the cell cannot divide before all chromosomes are aligned, but on the other hand, when they are ready, the separation is quite fast, lasting a few minutes. Our purpose is to use these opposed constraints to estimate the associated chemical rates. Results: To analyze the above constraints, we develop a markovian model to describe the dynamics of Cdc20 molecules. We compute the probability for no APC/C activation before time t, the distribution of Cdc20 at equilibrium and the mean time to complete APC/C activation after all chromosomes are attached. Conclusions: By studying Cdc20 inhibition and the activation time, we obtain a range for the main chemical reaction rates regulating the spindle assembly checkpoint and transition to anaphase.

Description: Gluten intolerance is a condition which affects an increasing percentage of the world's population and for which the only current treatment is a restrictive gluten free diet. However could the inclusion of a particular polysaccharide, or blends of different types, help with the provision of 'safer' foods for those individuals who suffer from this condition. We review the current knowledge on the prevalence, clinical symptoms and treatment of gluten intolerance, and the use and properties of the allergens responsible. We consider the potential for dietary fibre polysaccharides to sequester peptides that are responsible for activation of the disease in susceptible individuals, and consider the potential of co-sedimentation in the analytical ultracentrifuge as a molecular probe for finding interactions strong enough to be considered as useful.

Description: Background: In order to replicate within their cellular host, many viruses have developed self-assembly strategies for their capsids which are sufficiently robustas to be reconstituted in vitro. Mathematical models for virus self-assembly usually assume that the bonds leading to cluster formation have constant reactivity over the time course of assembly (direct assembly). In some cases, however, binding sites between the capsomers have been reported to be activated during the self-assembly process (hierarchical assembly). Results: In order to study possible advantages of suchhierarchical schemes for icosahedral virus capsid assembly, we useBrownian dynamics simulations of a patchy particle model that allowsus to switch binding sites on and off during assembly. For T1 viruses,we implement a hierarchical assembly scheme where inter-capsomer bondsbecome active only if a complete pentamer has been assembled. We finddirect assembly to be favorable for reversible bonds allowing forrepeated structural reorganizations, while hierarchical assembly isfavorable for strong bonds with small dissociation rate, as thissituation is less prone to kinetic trapping. However, at the same timeit is more vulnerable to monomer starvation during the finalphase. Increasing the number of initial monomers does have only a weakeffect on these general features. The differences between the twoassembly schemes become more pronounced for more complex virusgeometries, as shown here for T3 viruses, which assemble throughhomogeneous pentamers and heterogeneous hexamers in the hierarchicalscheme. In order to complement the simulations for this morecomplicated case, we introduce a master equation approach that agreeswell with the simulation results. Conclusions: Our analysis shows for which molecular parameters hierarchical assembly schemes can outperform direct ones. Hierarchical assembly is superior as it avoids kinetic trapping, but suffers more strongly from monomer starvation.These insights increase our physical understanding of an essential biological process, with many interesting potential applications in medicine and materials science.

Description: Background: Human carbonic anhydrases (CAs) play crucial role in various physiological processesincluding carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions,and various pathological processes, especially tumor progression. Therefore, CAs areinteresting targets for pharmaceutical research. The structure-activity relationships (SAR) ofdesigned inhibitors require detailed thermodynamic and structural characterization of thebinding reaction. Unfortunately, most publications list only the observed thermodynamicparameters that are significantly different from the intrinsic parameters. However, onlyintrinsic parameters could be used in the rational design and SAR of the novel compounds. Results: Intrinsic binding parameters for several inhibitors, including ethoxzolamide,trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIIIisozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy,entropy, and the heat capacity. They were determined by titration calorimetry and thermalshift assay in a wide pH and temperature range to dissect all linked protonation reactioncontributions. Conclusions: Precise determination of the inhibitor binding thermodynamics enabled correct intrinsicaffinity and enthalpy ranking of the compounds and provided the means for SAR analysis ofother rationally designed CA inhibitors.

Description: Background: G protein coupled receptors (GPCRs) are seven helical transmembrane proteins that function as signal transducers. They bind ligands in their extracellular and transmembrane regions and activate cognate G proteins at their intracellular surface at the other side of the membrane. The relay of allosteric communication between the ligand binding site and the distant G protein binding site is poorly understood. In this study, GREMLIN [1], a recently developed method that identifies networks of co-evolving residues from multiple sequence alignments, was used to identify those that may be involved in communicating the activation signal across the membrane. The GREMLIN-predicted long-range interactions between amino acids were analyzed with respect to the seven GPCR structures that have been crystallized at the time this study was undertaken. Results: GREMLIN significantly enriches the edges containing residues that are part of the ligand binding pocket, when compared to a control distribution of edges drawn from a random graph. An analysis of these edges reveals a minimal GPCR binding pocket containing four residues (T1183.33, M2075.42, Y2686.51 and A2927.39). Additionally, of the ten residues predicted to have the most long-range interactions (A1173.32, A2726.55, E1133.28, H2115.46, S186EC2, A2927.39, E1223.37, G902.57, G1143.29 and M2075.42), nine are part of the ligand binding pocket. Conclusions: We demonstrate the use of GREMLIN to reveal a network of statistically correlated and functionally important residues in class A GPCRs. GREMLIN identified that ligand binding pocket residues are extensively correlated with distal residues. An analysis of the GREMLIN edges across multiple structures suggests that there may be a minimal binding pocket common to the seven known GPCRs. Further, the activation of rhodopsin involves these long-range interactions between extracellular and intracellular domain residues mediated by the retinal domain.

Description: Background: EF-hand proteins can be activated by the binding of various heavy metals other than calcium,and such complexes can disturb the calcium-signaling pathway and cause toxicity and diseasecausing state. So far, no comprehensive study has been done to understand different heavymetals binding to calcium signaling proteins. Results: In this work, the flexibility of the EF-hand motifs are examined by crystallographic andthermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 fromEntamoeba histolytica (EhCaBP1). The structures of the EhCaBP1- heavy metal complexesare found to be overall similar, nevertheless specific differences in metal coordination, andsmall differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where twobariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirmthat EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the othermetals. These structures and thermodynamic studies reveal that the EF-hand motifs canaccommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are bothenthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sitesare simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinatewith water in this structure. For all the metals, binding to the 3rd site is only entropy driven. Conclusion: Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy.The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ boundstructure, except for the lack of water coordination. Sr2+ coordination seems to be a preformedin nature since all seven coordinating atoms are from the protein itself, which alsocorrelates with entropy contributions in Sr2+ binding. These findings improve ourunderstanding of metal association with calcium binding proteins and of metal inducedconformational changes.

Description: Background: Molecular Dynamics (MD) simulations are a promising tool to generate molecularunderstanding of processes related to the purification of proteins. Polyethylene glycols(PEG) of various length are commonly used in the production and purification of proteins.The molecular mechanisms behind PEG driven precipitation, aqueous two-phase formationor the effects of PEGylation are however still poorly understood. Results: In this paper, we ran MD simulations of single PEG molecules of variable length inexplicitly simulated water. The resulting structures are in good agreement withexperimentally determined 3D structures of PEG. The increase in surface hydrophobicity ofPEG of longer chain length could be explained on an atomic scale. PEG-water interactionsas well as aqueous two-phase formation in the presence of PO4 were found to be correlatedto PEG surface hydrophobicity. Conclusions: We were able to show that the taken MD simulation approach is capable of generating bothstructural data as well as molecule descriptors in agreement with experimental data. Thus,we are confident of having a good in silico representation of PEG.

Description: Background: Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results: We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 mum2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent alpha ~ 0.63 and an anomalous diffusion constant of Dalpha = 1.95 x 10-3 mum2/salpha. On a longer time scale (t 〉 ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 mum/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%. Conclusions: The mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.

Description: Background: Understanding the mechanical properties of chromatin is an essential step towards deciphering the physicalrules of gene regulation. In the past ten years, many single molecule experiments have been carried out, andhigh resolution measurements of the chromatin fiber stiffness are now available. Simulations have been usedin order to link those measurements with structural cues, but so far no clear agreement among different groupshas been reached. Results: We revisit here one of the most precise results obtained with carefully reconstituted fibers. Conclusions: We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by thestiffness of the DNA molecule and the 3D structure of the chromatin fiber.

Description: Background Accurate modeling of electrostatic potential and corresponding energies becomes increasingly important for understanding properties of biological macromolecules and their complexes. However, this is not an easy task due to the irregular shape of biological entities and the presence of water and mobile ions. Results Here we report a comprehensive suite for the well-known Poisson-Boltzmann solver, DelPhi, enriched with additional features to facilitate DelPhi usage. The suite allows for easy download of both DelPhi executable files and source code along with a makefile for local installations. The users can obtain the DelPhi manual and parameter files required for the corresponding investigation. Non-experienced researchers can download examples containing all necessary data to carry out DelPhi runs on a set of selected examples illustrating various DelPhi features and demonstrating DelPhi’s accuracy against analytical solutions. Conclusions DelPhi suite offers not only the DelPhi executable and sources files, examples and parameter files, but also provides links to third party developed resources either utilizing DelPhi or providing plugins for DelPhi. In addition, the users and developers are offered a forum to share ideas, resolve issues, report bugs and seek help with respect to the DelPhi package. The resource is available free of charge for academic users from URL: http://compbio.clemson.edu/DelPhi.php

Description: Background The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes. Results Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution. Conclusions We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge distribution contributes to the specific short-range interactions responsible for the formation of the bound complex. We also propose a scheme for coreceptor selectivity based on the sequence glycosylation motif, the 11/24/25 rule, and net charge.

Description: Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519–17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.

Description: Background The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature. Results In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount. Conclusions The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.

Description: Background G protein coupled receptors (GPCRs) are seven helical transmembrane proteins that function as signal transducers. They bind ligands in their extracellular and transmembrane regions and activate cognate G proteins at their intracellular surface at the other side of the membrane. The relay of allosteric communication between the ligand binding site and the distant G protein binding site is poorly understood. In this study, GREMLIN [1], a recently developed method that identifies networks of co-evolving residues from multiple sequence alignments, was used to identify those that may be involved in communicating the activation signal across the membrane. The GREMLIN-predicted long-range interactions between amino acids were analyzed with respect to the seven GPCR structures that have been crystallized at the time this study was undertaken. Results GREMLIN significantly enriches the edges containing residues that are part of the ligand binding pocket, when compared to a control distribution of edges drawn from a random graph. An analysis of these edges reveals a minimal GPCR binding pocket containing four residues (T1183.33, M2075.42, Y2686.51 and A2927.39). Additionally, of the ten residues predicted to have the most long-range interactions (A1173.32, A2726.55, E1133.28, H2115.46, S186EC2, A2927.39, E1223.37, G902.57, G1143.29 and M2075.42), nine are part of the ligand binding pocket. Conclusions We demonstrate the use of GREMLIN to reveal a network of statistically correlated and functionally important residues in class A GPCRs. GREMLIN identified that ligand binding pocket residues are extensively correlated with distal residues. An analysis of the GREMLIN edges across multiple structures suggests that there may be a minimal binding pocket common to the seven known GPCRs. Further, the activation of rhodopsin involves these long-range interactions between extracellular and intracellular domain residues mediated by the retinal domain.

Description: Background During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

Description: Background Bacteria dynamically regulate their intricate intracellular organization involving proteins that facilitate cell division, motility, and numerous other processes. Consistent with this sophisticated organization, bacteria are able to create asymmetries and spatial gradients of proteins by localizing signaling pathway components. We use mathematical modeling to investigate the biochemical and physical constraints on the generation of intracellular gradients by the asymmetric localization of a source and a sink. Results We present a systematic computational analysis of the effects of other regulatory mechanisms, such as synthesis, degradation, saturation, and cell growth. We also demonstrate that gradients can be established in a variety of bacterial morphologies such as rods, crescents, spheres, branched and constricted cells. Conclusions Taken together, these results suggest that gradients are a robust and potentially common mechanism for providing intracellular spatial cues.

Description: Background In order to replicate within their cellular host, many viruses have developed self-assembly strategies for their capsids which are sufficiently robust as to be reconstituted in vitro. Mathematical models for virus self-assembly usually assume that the bonds leading to cluster formation have constant reactivity over the time course of assembly (direct assembly). In some cases, however, binding sites between the capsomers have been reported to be activated during the self-assembly process (hierarchical assembly). Results In order to study possible advantages of such hierarchical schemes for icosahedral virus capsid assembly, we use Brownian dynamics simulations of a patchy particle model that allows us to switch binding sites on and off during assembly. For T1 viruses, we implement a hierarchical assembly scheme where inter-capsomer bonds become active only if a complete pentamer has been assembled. We find direct assembly to be favorable for reversible bonds allowing for repeated structural reorganizations, while hierarchical assembly is favorable for strong bonds with small dissociation rate, as this situation is less prone to kinetic trapping. However, at the same time it is more vulnerable to monomer starvation during the final phase. Increasing the number of initial monomers does have only a weak effect on these general features. The differences between the two assembly schemes become more pronounced for more complex virus geometries, as shown here for T3 viruses, which assemble through homogeneous pentamers and heterogeneous hexamers in the hierarchical scheme. In order to complement the simulations for this more complicated case, we introduce a master equation approach that agrees well with the simulation results. Conclusions Our analysis shows for which molecular parameters hierarchical assembly schemes can outperform direct ones and suggests that viruses with high bond stability might prefer hierarchical assembly schemes. These insights increase our physical understanding of an essential biological process, with many interesting potential applications in medicine and materials science.

Description: Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t 〉 ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%. Conclusions The mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.

Description: Background Understanding the mechanical properties of chromatin is an essential step towards deciphering the physical rules of gene regulation. In the past ten years, many single molecule experiments have been carried out, and high resolution measurements of the chromatin fiber stiffness are now available. Simulations have been used in order to link those measurements with structural cues, but so far no clear agreement among different groups has been reached. Results We revisit here some of the most precise experimental results obtained with carefully reconstituted fibers. Conclusions We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by the stiffness of the DNA molecule and the 3D structure of the chromatin fiber.

Description: Background The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces. Results We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated. Conclusions Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high-affinity interaction with ephrins because the weak interaction with small molecule was found to be insufficient to trigger the transition. Our results therefore highlight the key role of protein dynamics in Eph-ephrin signalling and would benefit future design of agonists/antagonists targeting Eph receptors.

Description: Background Cell migration plays an essential role in many biological processes, such as cancer metastasis, wound healing and immune response. Cell migration is mediated through protrusion and focal adhesion (FA) assembly, maturation and disassembly. Epidermal growth factor (EGF) is known to enhance migration rate in many cell types; however it is not known how FA maturation, FA dynamics and protrusion dynamics are regulated during EGF-induced migration. Here we use total internal reflection fluorescence (TIRF) microscopy and image analysis to quantify FA properties and protrusion dynamics under different doses of EGF stimulation. Results EGF was found to broaden the distribution of cell migration rates, generating more fast and slow cells. Furthermore, groups based on EGF stimulation condition or cell migration speed were marked by characteristic signatures. When data was binned based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was binned based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells. Conclusions Only a portion of the FA properties and protrusion dynamics that correlate with migration speed, correlate with EGF stimulation condition. Those that do not correlate with EGF stimulation condition constitute the most sensitive output for identifying why cells respond differently to EGF. The idea that EGF can both increase and decrease the migration speed of individual cells in a population has particular relevance to cancer metastasis where the microenvironment can select subpopulations based on some adhesion and protrusion characteristics, leading to a more invasive phenotype as would be seen if all cells responded like an “average” cell.

Description: Background EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins. Results In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1). The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven. Conclusion Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the protein itself, which also correlates with entropy contributions in Sr2+ binding. These findings improve our understanding of metal association with calcium binding proteins and of metal induced conformational changes.