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Apicomplexan parasites such as Toxoplasma gondii use gliding motility to infect host cells, and actin is involved in the regulation of this motility. However Toxoplasma actin, unlike vertebrate actin, does not form long conventional filaments and has been found to have very different polymerization properties. In this work in vitro experiments and molecular modeling are used to elucidate the molecular mechanism behind the unusual instability of parasite actin relative to muscle actin. In vitro observations suggested that the parasite actin are inherently unstable, and stable filaments were only able to be formed with high concentrations of phalloidin. Molecular models of parasite actin filament were constructed and the dynamics were compared with muscle filament actin dynamics to tease the out the differences at the molecular level between these two systems. We found specific amino acid differences gave rise to changes in the interactions between monomers within the filament, and mutation of these residues was able to restore filament stability in experiments. Implications for the structure of the actin filament and the interaction of actin binding drugs will be discussed.

The mechanical properties of microtubules have been extensively studied using a wide range of biophysical techniques, seeking to understand the mechanics of these cylindrical polymers. Here we develop a method for connecting all-atom molecular dynamics simulations with continuum mechanics and show how this can be applied to understand microtubule mechanics. Our coarse-graining technique applied to the microscopic simulation system yields consistent predictions for the Young’s modulus and persistence length of microtubules, while clearly demonstrating how binding of the drug Taxol decreases the stiffness of microtubules. The techniques we develop should be widely applicable to other macromolecular systems. Microtubules are essential players in a wide range of cellular functions. The protein tubulin polymerizes to form these filaments resulting in a hollow cylinder with a diameter of about 25 nm and lengths that range from tens of nanometers to tens of microns. Microtubules are the most rigid structures within the cell and their mechanical stability is particularly important for the role that microtubules play in cell division, cellular transport, and their basic ability to help provide cell shape. The mechanical properties of purified microtubules have been studied extensively using many biophysical techniques, including atomic force microscopy, thermal bending, and single molecule techniques [1–11]. Since microtubules are dynamic polymers and have intrinsic instability, many of these experimental studies have been performed on Taxol-stabilized microtubules, although the mechanical consequences of the drug Taxol for microtubule mechanics remains unclear. The majority of studies have found that the addition of Taxol results in ‘‘softer’’ microtubules with a shorter persistence length and lower Young’s modulus [1–5], while one published study had found the opposite result [6]. Recently, even the standard elastic model of microtubules characterized by a single elastic bending constant has been called into question by reports of length-dependent stiffness [12]. Thus, in spite of decades of experimental study, many basic questions and puzzles remain concerning the mechanics of even individual microtubules in vitro, let alone in vivo. Here, we combine all-atom simulations and continuum elastic modeling in order to determine both the elastic parameters of persistence length and Young’s modulus, as well as to test the applicability of simple, continuum shell elasticity for microtubules. Based on a molecular dynamics simulation of a small section of six tubulin monomers [13], we show that the observed fluctuations of this section are consistent with linear elastic constants for both bending and stretching along the microtubule axis. From these measurements, we determine the effective elastic properties for the microtubule wall, treated as an elastic sheet. With the addition of Taxol, we find increased flexibility and reduced bending and stretching moduli. We then examine the shear strain fluctuations of the tubulin sheet and find evidence for enhanced shear compliance, relative to the predictions of homogenous and isotropic elasticity. While anomalously large shear compliance has been suggested recently in connection with lengthdependent bending stiffness of microtubules [12], we find a much smaller degree of anisotropy than previously suggested. The shear elasticity we find is closer to recent experiments [9].

Molecular modeling techniques have made significant advances in recent years and are becoming essential components of many chemical, physical and biological studies. Here we present three widely used techniques used in the simulation of biomolecular systems: structural and homology modeling, molecular dynamics and molecular docking. For each of these topics we present a brief discussion of the underlying scientific basis of the technique, some simple examples of how the method is commonly applied, and some discussion of the limitations and caveats of which the user should be aware. References for further reading as well as an extensive list of software resources are provided.

The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.

Ion-channel function is determined by its gating movement. Yet, molecular dynamics and electrophysiological simulations were never combined to link molecular structure to function. We performed multiscale molecular dynamics and continuum electrostatics calculations to simulate a cardiac K + channel (I Ks ) gating and its alteration by mutations that cause arrhythmias and sudden death. An all-atom model of the I Ks α-subunit KCNQ1, based on the recent Kv1.2 structure, is used to calculate electrostatic energies during gating. Simulations are compared with experiments where varying degrees of positive charge—added via point mutation—progressively reduce current. Whole-cell simulations show that mutations cause action potential and ECG QT interval prolongation, consistent with clinical phenotypes. This framework allows integration of multiscale observations to study the molecular basis of excitation and its alteration by disease.

Actin,a component of the cytoskeletal system, is a polymer that is critical for maintaining the shape and motility of a cell. This is achieved by a complex dynamic regulation of rapid polymerization and depolymerization of actin. As part of its defense mechanism, certain species of fungi and marine sponges produces cyclic peptide compounds like Jasplakinolide and Phalloidin that interferes with the actin depolymerization in foreign species. In this work we use computational methods like molecular dynamics, docking and QM/MM with to elucidate the molecular details of the interaction of these compounds with mammalian and parasite actin filament. Our analysis, in addition with experimental observations from our collaborators, also helps us to propose possible mechanisms for the polymer stabilizing property of these compounds.

Calcium plays a major role in controlling the opening and closing of the large conductance BKCa channels. Two high affinity binding sites have been identified in the channel structure and one of these sites is the DRDD loop in the N-terminus of the RCK1 domain. Mutation of the first aspartate in this conserved DRDD motif significantly reduces Ca2+ sensitivity and hence this residue has been implicated as a coordinating group in the binding site. Here we present results on the prediction of the Ca2+ binding site based on a series of detailed computational studies. The basic protocol involves multiple iterations of random ion placement, implicit solvent molecular dynamics simulations and statistical analysis. Our resulting model matches very well with existing mutagenesis data, and subsequent explicit solvent molecular dynamics simulations have been performed using this Ca2+ bound structure. Comparison of the dynamics and conformations of the Ca2+ bound and unbound simulations reveal a concerted conformational change in the structure and suggest a potential mechanism for calcium dependent activation of these channels.

Molecular modeling techniques have made significant advances in recent years and are becoming essential components of many chemical, physical and biological studies. Here we present three widely used techniques used in the simulation of biomolecular systems: structural and homology modeling, molecular dynamics and molecular docking. For each of these topics we present a brief discussion of the underlying scientific basis of the technique, some simple examples of how the method is commonly applied, and some discussion of the limitations and caveats of which the user should be aware. References for further reading as well as an extensive list of software resources are provided.

Contrast agents targeted to molecular markers of disease are currently being developed with the goal of identifying disease early and evaluating treatment effectiveness using noninvasive imaging modalities such as MRI. Pharmacokinetic profiling of the binding of targeted contrast agents, while theoretically possible with MRI, has thus far only been demonstrated with more sensitive imaging techniques. Paramagnetic liquid perfluorocarbon nanoparticles were formulated to target alpha(v)beta(3)-integrins associated with early atherosclerosis in cholesterol-fed rabbits to produce a measurable signal increase on magnetic resonance images after binding. In this work, we combine quantitative information of the in vivo binding of this agent over time obtained by means of MRI with blood sampling to derive pharmacokinetic parameters using simultaneous and individual fitting of the data to a three compartment model. A doubling of tissue exposure (or area under the curve) is obtained with targeted as compared to control nanoparticles, and key parameter differences are discovered that may aid in development of models for targeted drug delivery.

Cardiovascular molecular imaging with targeted nanoparticles has emerged as a promising method for early detection of atherosclerosis and vulnerable plaque. However, traditional pharmacokinetic models for diffusible drugs are inadequate to describe the efficacy of nanoparticle carriers of diagnostic and therapeutic cargos. To quantify blood and MRI tissue signals from gadolinium-loaded nanoparticles (Gd-NP) targeted to avb3 integrins expressed on angiogenic capillaries in the aortas of cholesterol-fed rabbit, a novel 4 compartment open PK model was developed that utilized a unique simultaneous fitting scheme. In 10 rabbits fed 0.25% cholesterol for 3 months, the concentration of nanoparticles was measured serially from blood samples after injection of 1 ml/kg of targeted or nontargeted Gd-NP. The MRI proton signatures emanating from nanoparticles bound to the expanded vasa vasorum of the descending thoracic aorta was computed for all aortic cross sections in 1.5T T1w fa-suppressed spin-echo images. Based on the PK analysis, the concentration of targeted nanoparticles in the aortic wall is double that of non-targeted nanoparticles. Notably, this signal enhancement is achieved with 20x less gadolinium (4.6x10 −3 mmol Gd/kg BW) as compared with the dose of conventional gadolinium agents (0.1 mmol/kg). Further, the PK analysis shows that the targeted nanoparticles are more than three times more effective at reaching the aortic wall. These results should facilitate development and use of nanotechnologies intended for early detection of atherosclerosis and provide enhanced understanding of the kinetics and mechanisms of active targeting of plaque angiogenesis.

The heterodimeric actin capping protein, referred to here as “CP,” is an essential element of the actin cytoskeleton, binding to the barbed ends of actin filaments and regulating their polymerization. In vitro, CP has a critical role in the dendritic nucleation process of actin assembly mediated by Arp2/3 complex, and in vivo, CP is important for actin assembly and actin-based process of morphogenesis and differentiation. Recent studies have provided new insight into the mechanism of CP binding the barbed end, which raises new possibilities for the dynamics of CP and actin in cells. In addition, a number of molecules that bind and regulate CP have been discovered, suggesting new ideas for how CP may integrate into diverse processes of cell physiology.

Molecular modeling techniques have truly come of age in recent decades, and here we cover several of the most commonly used techniques, namely molecular dynamics, Brownian dynamics, and molecular docking. In each case, we explain the physical basis and limitations of the various techniques and then illustrate their application to various problems related to the cytoskeleton. This set of studies covers a relatively wide range of examples and is comprehensive enough to clearly see how these techniques could be applied to other systems. Finally, we cover several related methodologies that expand on these basic techniques to allow for more detailed and specific simulation and analysis.

Mathematical modeling has become increasingly important in many areas of biology during the past two decades, and the area of cell migration and motility has seen significant contributions from a wide range of modeling approaches. In this chapter, we cover examples from the broad range of work in this area, emphasizing the models' biological significance and the relationships between them. We focus on three specific areas: cell protrusion, cell adhesion, and retraction/whole-cell models. At the end of this chapter, we provide our perspective on issues that future models and experiments should consider in order to advance the boundaries of this field.

Molecular modeling techniques have truly come of age in recent decades, and here we cover several of the most commonly used techniques, namely molecular dynamics, Brownian dynamics, and molecular docking. In each case, we explain the physical basis and limitations of the various techniques and then illustrate their application to various problems related to the cytoskeleton. This set of studies covers a relatively wide range of examples and is comprehensive enough to clearly see how these techniques could be applied to other systems. Finally, we cover several related methodologies that expand on these basic techniques to allow for more detailed and specific simulation and analysis.

Mathematical modeling has become increasingly important in many areas of biology during the past two decades, and the area of cell migration and motility has seen significant contributions from a wide range of modeling approaches. In this chapter, we cover examples from the broad range of work in this area, emphasizing the models' biological significance and the relationships between them. We focus on three specific areas: cell protrusion, cell adhesion, and retraction/whole-cell models. At the end of this chapter, we provide our perspective on issues that future models and experiments should consider in order to advance the boundaries of this field.

International audience; Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 A) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.

Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (C(c)) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP- and ADP-actin polymerization and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the C(c) of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (approximately 30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.

Adenosine 5′-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5′-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an α-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.

The escape of a diffusing particle from a potential well is an important aspect of many dynamic processes in chemistry, physics, and biology, and such an escape process often involves finding a restricted region or patch in a multidimensional potential well. We study an idealized model of this process via simulation and analytic theory. By combining results from special cases having either high symmetry or zero potential, we obtain a simple formula for the first-contact time for a particle moving to a boundary patch in an arbitrary number of dimensions. We apply this formula in two, three, and six dimensions. The predicted dependences of the first-contact time on the well depth and patch size are compared to results from simulations, and close agreement is found. We extend the theory to calculate the first-contact time between two particles in separate harmonic potential wells. As an application of this extended theory, we calculate the first-contact time for two parallel semiflexible biopolymer filaments and compare these results to previous simulations.

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP2), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP2in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.

Despite years of use as commercial herbicides, it is still unclear how dinitroanilines interact with tubulin, how they cause microtubule disassembly, and why they are selectively active against plant and protozoan tubulin. In this work, through a series of computational studies, a common binding site of oryzalin, trifluralin, and GB-II-5 on apicomplexan and kinetoplastid alpha-tubulin is proposed. Furthermore, to investigate how dinitroanilines affect tubulin dynamics, molecular dynamics simulations of Leishmania alpha-tubulin with and without a bound dinitroaniline are performed. The results obtained provide insight into the molecular mechanism by which these compounds interact with tubulin and function to prevent microtubule assembly. Finally, to aid in the design of effective parasitic microtubule inhibitors, several novel dinitroaniline analogues are evaluated. The location of the binding site and the relative binding affinities of the dinitroanilines all agree well with experimental data.

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a Kd of 10-50 nm. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP beta subunit was important for binding V-1, with lesser contributions from the alpha subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1.CP complex had no capping activity. Two loops of V-1, which extend out from the alpha-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the beta tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the alpha1 and alpha2 isoforms as well as the mutant lacking the alpha tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile beta-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.

Microtubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left-right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large-scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.

The availability of an atomically-detailed tubulin structure has created new possibilities for investigating many features of microtubule structure and function. Through the use of a newly developed method for large scale electrostatic calculations, we are able to explain several key microtubule properties. This talk will present recent results on microtubule stability, plus and minus end polymerization, as well as the physical basis for the observed helix and seam.

The role of electrostatic interactions and ionic strength effects in actin polymerization has been well documented. This behavior has been explained using Brownian dynamics simulations where the electrostatics were based on Poisson-Boltzmann calculations. In cases where the electrostatic potential is strong, the assumption of Boltzmann distributed ions become inaccurate since it ignores the finite size and interactions of the ions. Here I present a new method for including explicit counterions in electrostatic calculations and show how these influence actin polymerization and protein interactions in general.

Summary The ‘GTP cap' of the microtubule has long been postulated to exist, but a recent experiment gives us the first quantitative measurements of the cap size in the cell.

Advances in mathematics and computer technology, together with advances in structural biology, are opening the way to detailed modeling of biology at the molecular and cellular levels. One objective of such studies is the development of a more complete understanding of biological systems, including the emergence of behavior at the cellular level from that at the molecular level. Another objective is the development of more sophisticated models for structure-aided discovery of new pharmaceuticals.

Nature Reviews Molecular Cell Biology 15, 677–689 (2014) On page 684 of the above article, there was a mistake in Figure 3b: the PH domain of CARMIL preferentially binds to monophosphorylated membrane lipids rather than to PtdIns(3)P, PtdIns(3,4)P2 or PtdIns(3,4,5)P3. This has been corrected online.We apologize for any confusion caused to readers.

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.

Calcium plays a major role in controlling the opening and closing of the large conductance BKCa channels. Two high affinity binding sites have been identified in the channel structure and one of these sites is the DRDD loop in the N-terminus of the RCK1 domain. Mutation of the first aspartate in this conserved DRDD motif significantly reduces Ca2+ sensitivity and hence this residue has been implicated as a coordinating group in the binding site. Here we present results on the prediction of the Ca2+ binding site based on a series of detailed computational studies. We use a novel multisite cation model for calcium ion to accurately simulate the ion-coordination. The basic protocol involves multiple iterations of random ion placement, implicit solvent molecular dynamics simulations and statistical analysis. Our resulting model matches very well with existing mutagenesis data, and subsequent explicit solvent molecular dynamics simulations have been performed using this Ca2+ bound structure. Comparison of the dynamics and conformations of the Ca2+ bound and unbound simulations reveal a concerted conformational change in the structure and suggest a potential mechanism for calcium dependent activation of these channels.

Voltage and Ca2+ activated BK channels modulate neuronal activities. Previous studies found that Ca2+ binding sites and the activation gate are spatially separated, but how Ca2+ binding couples to gate opening is not clear. We address this question by studying how a mutation in BK channels, which is associated with generalized epilepsy and paroxysmal dyskinesia (D434G in hSlo, the epilepsy mutation), enhances Ca2+ sensitivity. The epilepsy mutation is located in a structural domain (the AC region) that connects the S6 transmembrane segment with the cytoplasmic domain and close to a putative Ca2+ binding site. Mutagenesis studies show that the AC region is important in the coupling between Ca2+ binding and gate opening. We found that discrete locations in the AC region that are distant from the epilepsy mutation alter the functional effects of the mutation. Thus, the epilepsy mutation enhances Ca2+ sensitivity by an allosteric mechanism affecting the coupling between Ca2+ binding and gate opening. Interestingly, mutating the Asp residue to amino acids other than Gly does not result in the same phenotype as the epilepsy mutation, suggesting that a change in protein flexibility is involved in the effects of the mutation. Consistent with this result, altering the flexibility of the channel protein by changing viscosity of the intracellular solution also modulates Ca2+ sensitivity; and the epilepsy mutation reduces such modulation, possibly because it has already altered the flexibility. These results are consistent with a model that the peptide loop where the putative Ca2+ binding site and the epilepsy mutation are located acts as a spring-hinge for the conformational change of the AC region during channel opening; Ca2+ binding and the epilepsy mutation affect channel gating by altering the function of this spring-hinge.

Identifying inhibitors of protein–protein interactions is an ongoing challenge in the field of drug design, but the use of peptide fragments based on a known binding interface is showing promise.

The regulation of actin polymerization within the cell is critical for many cell functions. Cofilin plays an important part in this process since it binds and severs actin filaments leading to depolymerization as well as the creation of new barbed ends. Although the details of cofilin's interaction with G-actin have been elucidated through a range of experimental studies, the specific interactions with F-actin have remained more elusive. Here we present the results of a detailed computational study involving a combination of molecular dynamics simulations and protein-protein docking. The resulting structural model for the cofilin/F-actin complex matches very well with existing cryoEM and mutagenesis data. Further insight is gained from a sequence and secondary structure alignment of cofilin analogs that strongly supports our binding model. The implications of this binding model in the function and severing action of cofilin are discussed.

We measured the lengths of actin filaments formed by spontaneous polymerization of highly purified actin monomers by fluorescence microscopy after labeling with rhodamine-phalloidin. The length distributions are exponential with a mean of approximately 7 microm (2600 subunits). This length is independent of the initial concentration of actin monomer, an observation inconsistent with a simple nucleation-elongation mechanism. However, with the addition of physically reasonable rates of filament annealing and fragmenting, a nucleation-elongation mechanism can reproduce the observed average length of filaments in two types of experiments: 1) filaments formed from a wide range of highly purified actin monomer concentrations, and 2) filaments formed from 24 microM actin over a range of CapZ concentrations.

The cytoskeleton of eukaryotic cells contains networks of protein polymers called microtubules which structurally and functionally organize their interiors. Both in vivo and in vitro microtubules exhibit a fascinating and yet poorly understood array of important functions involving complex self-organization phenomena which are very sensitive to physiological and laboratory conditions, respectively. In this paper we discuss the main physical characteristics of microtubules focusing our attention on four particular aspects: (a) the dynamics of their assembly and disassembly processes (b) the types and the range of existence of ordered dipolar phases and (c) modes of energy transfer and (d) information processing capabilities.

The protozoan parasite Toxoplasma gondii relies on actin polymerization for motility and host cell invasion. Despite this strict requirement for actin filaments, T. gondii actin (TgACTI) remains largely unpolymerized in vivo and recombinant TgACTI only forms short filaments in vitro. TgACTI has several amino acid substitutions when compared with muscle actin, and our previous work showed that these changes significantly diminish the lateral contacts and interactions within the actin filament. The net result is that the two strands of the filament assemble with less cooperativity and not surprisingly this has significant effects on the polymerization kinetics. Here we show that the highly unusual characteristics of TgACTI result from isodesmic polymerization rather than the nucleation-elongation kinetics of conventional eukaryotic actins. TgACTI polymerization kinetics lack a lag phase and critical concentration, and all of the results from dynamic light scattering, dilution induced depolymerization, and sedimentation can be fit using a single pair of kinetic rate constants. These findings expand the repertoire of how actin functions in cell motility and offer clues about the evolution of self-assembling, stabilized protein polymers.

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here we re-examine the polymerization properties of actin in Toxoplasma gondii, unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. Polymerization kinetics of actin in T. gondii lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly and the size distribution of actin filaments in T. gondii in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers.

Interiors of living cells are structurally and dynamically organized by networks of protein polymers called the cytoskeleton. Of these filamentous structures microtubules are best characterized and appear to be most fundamental. Bulk cytoplasm of living eu-kariotic cells contains parallel-networks of individual microtubules which are interconnected by filamentous strands (so called MAPs or microtubule associated proteins). In this paper we report on the main physical features of microtubules focusing our attention on the microtubules dynamics, assembly (disassembly), nonlinear modes suitable for signaling and the spin-glass phase which is ideal for information processing.

Current ion models in molecular mechanics are simple spheres, and their interactions are solely determined from the radius of the sphere and the total charge. This set of parameters is chosen to closely reproduce the hydration free energy for the ion, but this exercise uses all the available degrees of freedom and our ability to reproduce the binding free energy to a protein or other thermodynamic quantities is therefore limited. Here we introduce a new model where we distribute the total charge of the ion into n-dummy centers that are placed in the direction of the coordinating atoms. We have parameterized this model for two divalent cations, Ca2+ and Mg2+, and have tested the model’s accuracy in a variety of simulations. With this model we are not only able to correctly predict the free energy and selectivity for cation binding sites, but we achieve better coordination geometries and can capture more subtle effects such as the exchange of inner shell waters. One further advantage of this model is that it does not use higher-order electrostatics and thus can be easily used with standard force fields.

Taxol is a commonly used antitumor agent that hyperstabilizes microtubules and prevents cell division. The interaction of Taxol with tubulin and the microtubule has been studied through a wide array of experimental techniques; however, the exact molecular mechanism by which Taxol stabilizes microtubules has remained elusive. In this study, through the use of large-scale molecular simulations, we show that Taxol affects the interactions between the M and H1-S2 loops of adjacent tubulin dimers leading to more stable interprotofilament interactions. More importantly, we demonstrate that Taxol binding leads to a significant increase in the dynamics and flexibility of the portion of β-tubulin that surrounds the bound nucleotide and makes contact with the α-monomer of the next dimer in the protofilament. We conclude that this increase in flexibility allows the microtubule to counteract the conformational changes induced by nucleotide hydrolysis and keeps the protofilaments in a straight conformation, resulting in a stable microtubule.

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.

Summary Background Microtubules (MTs) support diverse transport and force generation processes in cells. Both α- and β-tubulin proteins possess carboxy-terminal tail regions (CTTs) that are negatively charged, intrinsically disordered, and project from the MT surface where they interact with motors and other proteins. Although CTTs are presumed to play important roles in MT networks, these roles have not been determined in vivo. Results We examined the function of CTTs in vivo by using a systematic collection of mutants in budding yeast. We find that CTTs are not essential; however, loss of either α- or β-CTT sensitizes cells to MT-destabilizing drugs. β-CTT, but not α-CTT, regulates MT dynamics by increasing frequencies of catastrophe and rescue events. In addition, β-CTT is critical for the assembly of the mitotic spindle and its elongation during anaphase. We use genome-wide genetic interaction screens to identify roles for α- and β-CTTs, including a specific role for β-CTT in supporting kinesin-5/Cin8. Our genetic screens also identified novel interactions with pathways not related to canonical MT functions. Conclusions We conclude that α- and β-CTTs play important and largely discrete roles in MT networks. β-CTT promotes MT dynamics. β-CTT also regulates force generation in the mitotic spindle by supporting kinesin-5/Cin8 and dampening dynein. Our genetic screens identify links between α- and β-CTT and additional cellular pathways and suggest novel functions.

The dynamic assembly of microtubules is a key factor in many of their functions in the cell and recent experiments give new insight into this process at the molecular level.