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Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.
We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in nonpocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu.
Lipid–polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle–membrane engagement, followed by a slow growth in the number of ligand–receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand–receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle–membrane interactions to occur even when the ligand density is low. The LPH–membrane avidity is enhanced by the increased stability of each receptor–ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle–cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.
Linactants, molecules that preferentially localize at the boundary of lipid membrane domains, are attracting considerable attention in recent years due to the recognition that they might regulate lipid-phase separation and thereby modulate membrane morphology. Recent studies have also shown that clustering of some line active agents enhances their ability to modulate membrane curvature. However, the molecular origin of this phenomenon, and the degree to which it impacts biological membranes, remains poorly understood. In this work, we have investigated how linactants induce shape change in multidomain small unilamallar vesicles (SUVs) using extensive dissipative particle dynamics simulations. The linactant was modeled as a two-tailed hybrid lipid with the two tails differing in preference for different lipid domains. We found that addition of a small amount of linactants (∼1%) to a two-domain vesicle leads to substantial reduction in the line tension and neck curvature at the domain boundary. Using cross-linking as a surrogate for clustering, we further show that linactant clusters substantially enhance the boundary preference and therefore the reduction in neck curvature. Moreover, on the basis of analyses of the corresponding changes in the membrane energetics, we highlight how linactants might stabilize nanoscale domains. These results have important implication
The mechanism of curvature generation in membranes has been studied for decades due to its important role in many cellular functions. However, it is not clear if, or how, aggregates of lipid-anchored proteins might affect the geometry and elastic property of membranes. As an initial step toward addressing this issue, we performed structural, geometrical, and stress field analyses of coarse-grained molecular dynamics trajectories of a domain-forming bilayer in which an aggregate of lipidated proteins was asymmetrically bound. The results suggest a general mechanism whereby asymmetric incorporation of lipid-modified protein aggregates curve multidomain membranes primarily by expanding the surface area of the monolayer in which the lipid anchor is inserted.
Over the last 40 years, we have learnt a great deal about the Ras onco-proteins. These intracellular molecular switches are essential for the function of a variety of physiological processes, including signal transduction cascades responsible for cell growth and proliferation. Molecular simulations and free energy calculations have played an essential role in elucidating the conformational dynamics and energetics underlying the GTP hydrolysis reaction catalysed by Ras. Here we present an overview of the main lessons from molecular simulations on the GTPase reaction and conformational dynamics of this important anti-cancer drug target. In the first part, we summarise insights from quantum mechanical and combined quantum mechanical/molecular mechanical simulations as well as other free energy methods and highlight consensus viewpoints as well as remaining controversies. The second part provides a very brief overview of new insights emerging from large-scale molecular dynamics simulations. We conclude with a perspective regarding future studies of Ras where computational approaches will likely play an active role.
Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor.
Acid-sensing ion channels are cation channels activated by external protons and play roles in nociception, synaptic transmission, and the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding, there is a conformational change that increases LRET efficiency between the probes at the thumb and finger subdomains in the extracellular domain of acid-sensing ion channels. Additionally, we show that this conformational change is lost upon mutating Asp-238, Glu-239, and Asp-260, which line the finger domains, to alanines. Electrophysiological studies showed that the single mutant D260A shifted the EC50 by 0.2 pH units, the double mutant D238A/E239A shifted the EC50 by 2.5 pH units, and the triple mutant D238A/E239A/D260A exhibited no response to protons despite surface expression. The LRET experiments on D238A/E239A/D260A showed no changes in LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which are involved in carboxyl/carboxylate interactions, are essential for proton-induced conformational changes in the extracellular domain, which in turn are necessary for receptor activation.
Ras proteins are attached to the inner leaflet of the plasma membrane via a lipid-modified anchor. Membrane-bound Ras proteins laterally segregate into nanoscale signaling platforms called nanoclusters. It has been shown that the membrane domain preference of Ras nanoclusters varies with the nature of lipidation but their effect on the membrane has not been well understood. To investigate the effect of Ras insertion on the membrane structure, we carried out numerous coarse-grained molecular dynamics (CGMD) simulations on a two-domain DPPC/DLiPC/cholesterol lipid bilayer in which different numbers and types of H-Ras peptides were attached on one side. We have shown previously that this lipid mixture forms co-existing liquid-ordered/liquid-disordered (Lo/Ld) domains and that different H-Ras peptides form clusters that variously accumulate in the Lo/Ld regions or the boundary between them. Here we show that asymmetric insertion of each of these peptides induces a vertical relative displacement of the domains and deforms the bilayer, with the domain boundary serving as the center of deformation. The extent of deformation, however, varies with the type and number of lipid modifications which determine the degree to which the stress caused by asymmetric peptide insertion is relieved by inter-leaflet cholesterol transfer and lipid tilt. In addition, we have characterized the mechanism of bilayer deformation based on the collective effect of the Ras peptides on the inter-leaflet surface area, pressure profile and line tension differences. This allowed us to elucidate how Ras lipid modification affects membrane geometry and how a two-domain bilayer adjusts its shape through boundary deformation. The result contributes to a better understanding of Ras signaling platforms and highlights some of the mechanisms by which a multi-domain membrane responds to external perturbation.
Prolonged usage of nonsteroidal anti-inflammatory drugs (NSAIDs) causes gastrointestinal injury. Bile acids and phospholipids have been shown to exasperate and attenuate NSAIDs' toxicity, respectively. However, the molecular mechanisms underlying these effects remain undetermined. We have investigated the molecular interactions in various mixtures of indomethacin (Indo), a commonly used NSAID, and cholic acid (CA), a bile acid, in the presence and absence of palmitoyloleylphosphatidylcholine (POPC) lipids. We found that CA and Indo spontaneously form mixed micelles, with the hydrophobic face of CA and hydrophobic region of Indo forming the core. Increasing the Indo concentration resulted in more stable and larger aggregates that contain a progressively larger number of Indo molecules. More dynamic aggregates with a maximum size of 15 were obtained when the relative concentration of CA was higher. The mixture of CA, Indo, and POPC also led to ternary mixed micelles in which CA and Indo distribute almost uniformly on the surface such that intra-CA, intra-Indo, and CA/Indo interactions are minimized. A number of previous reports have shown that Indo perforates the cell membrane in the presence of bile acids (e.g., Petruzzelli et al., (2006) Dig. Dis. Sci., 51, 766–774). We propose that this may be related to the stable, highly charged, large CA/Indo binary micelles observed in our simulations. Similarly, the diminished ability of the CA/Indo mixture to aggregate in the presence of POPC may partly explain the lower toxicity of PC-conjugated NSAIDs.
Nine membrane-bound adenylyl cyclase (AC) isoforms catalyze the production of the second messenger cyclic AMP (cAMP) in response to various stimuli. Reduction of AC activity has well documented benefits, including benefits for heart disease and pain. These roles have inspired development of isoform-selective AC inhibitors, a lack of which currently limits exploration of functions and/or treatment of dysfunctions involving AC/cAMP signaling. However, inhibitors described as AC5- or AC1-selective have not been screened against the full panel of AC isoforms. We have measured pharmacological inhibitor profiles for all transmembrane AC isoforms. We found that 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536), 2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), and adenine 9-β-d-arabinofuranoside (Ara-A), described as supposedly AC5-selective, do not discriminate between AC5 and AC6, whereas the putative AC1-selective inhibitor 5-[[2-(6-amino-9H-purin-9-yl)ethyl]amino]-1-pentanol (NB001) does not directly target AC1 to reduce cAMP levels. A structure-based virtual screen targeting the ATP binding site of AC was used to identify novel chemical structures that show some preference for AC1 or AC2. Mutation of the AC2 forskolin binding pocket does not interfere with inhibition by SQ22,536 or the novel AC2 inhibitor, suggesting binding to the catalytic site. Thus, we show that compounds lacking the adenine chemical signature and targeting the ATP binding site can potentially be used to develop AC isoform-specific inhibitors, and discuss the need to reinterpret literature using AC5/6-selective molecules SQ22,536, NKY80, and Ara-A.
Lipid-anchored Ras oncoproteins assemble into transient, nano-sized substructures on the plasma membrane. These substructures, called nanoclusters, were proposed to be crucial for high-fidelity signal transmission in cells. However, the molecular basis of Ras nanoclustering is poorly understood. In this work, we used coarse-grained (CG) molecular dynamics simulations to investigate the molecular mechanism by which full-length H-ras proteins form nanoclusters in a model membrane. We chose two different conformations of H-ras that were proposed to represent the active and inactive state of the protein, and a domain-forming model bilayer made up of di16:0-PC (DPPC), di18:2-PC (DLiPC) and cholesterol. We found that, irrespective of the initial conformation, Ras molecules assembled into a single large aggregate. However, the two binding modes, which are characterized by the different orientation of the G-domain with respect to the membrane, differ in dynamics and organization during and after aggregation. Some of these differences involve regions of Ras that are important for effector/modulator binding, which may partly explain observed differences in the ability of active and inactive H-ras nanoclusters to recruit effectors. The simulations also revealed some limitations in the CG force field to study protein assembly in solution, which we discuss in the context of proposed potential avenues of improvement.
A great deal has been learned over the last several decades about the function of Ras proteins in solution and membrane environments. While much of this knowledge has been derived from a plethora of experimental techniques, computer simulations have also played a substantial role.Our goal here is to summarize the contribution of molecular simulations to our current understanding of normal and aberrant Ras function. We focus on lessons from molecular dynamics simulations in aqueous and membrane environments.The central message is that a close interaction between theory and simulation on the one hand and cell-biological, spectroscopic and other experimental approaches on the other has played, and will likely continue to play, a vital role in Ras research.Atomistic insights emerging from detailed simulations of Ras in solution and in bilayers may be the key to unlock the secret that to date prevented development of selective anti-Ras inhibitors for cancer therapy. Molecular dynamics; Advanced simulation; Protein motion; Membrane binding; Clustering; Oncogenic Ras
Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)—a bicyclic diterpenoid lactone isolated from Andrographis paniculata—and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP–GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.
Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.
Experiments have shown that homologous Ras proteins containing different lipid modification, which is required for membrane binding, form nonoverlapping nanoclusters on the plasma membrane. However, the physical basis for clustering and lateral organization remains poorly understood. We have begun to tackle this issue using coarse-grained molecular dynamics simulations of the H-ras lipid anchor (tH), a triply lipid-modified heptapeptide embedded in a domain-forming mixed lipid bilayer [Janosi L. et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 8097]. Here we use the same simulation approach to investigate the effect of peptide concentration and bilayer composition on the clustering and lateral distribution of tH. We found no major difference in the clustering behavior of tH above a certain concentration. However, the simulations predict the existence of a critical concentration below which tH does not form nanoclusters. Moreover, our data demonstrate that cholesterol enhances the stability of tH nanoclusters but is not required for their formation. Finally, analyses of peptide distributions and partition free energies allowed us to quantitatively describe how clustering facilitates the accumulation of tH at the interface between ordered and disordered domains of the simulated bilayer systems. These thermodynamic insights represent some of the key elements for a comprehensive understanding of the molecular basis for the formation and stability of Ras signaling platforms.
The dynamic assembly and lateral organization of Ras proteins on the plasma membrane has been the focus of much research in recent years. It has been shown that different isoforms of Ras proteins share a nearly identical catalytic domain, yet form distinct and non-overlapping nanoclusters. Though this difference in the clustering behavior of Ras proteins has been attributed largely to their different C terminal lipid modification, its precise physical basis was not determined. Recently, we used computer simulations to study the mechanism by which the triply lipid-modified membrane-anchor of H-ras, and its partially de-lipidated variants, form nanoclusters in a model lipid bilayer. We found that the specific nature of the lipid modification is less important for cluster formation, but plays a key role for the domain-specific distribution of the nanoclusters. Here we provide additional details on the interplay between bilayer structure perturbation and peptide-peptide association that provide the physical driving force for clustering. We present some thoughts about how enthalpic (i.e., interaction) and entropic effects might regulate nanocluster size and stability.
Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells.
Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID–BA or NSAID–BA–PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA–IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA–IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA–IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA–IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.
Recent experiments have shown that membrane-bound Ras proteins form transient, nanoscale signaling platforms that play a crucial role in high-fidelity signal transmission. However, a detailed characterization of these dynamic proteolipid substructures by high-resolution experimental techniques remains elusive. Here we use extensive semiatomic simulations to reveal the molecular basis for the formation and domain-specific distribution of Ras nanoclusters. As model systems, we chose the triply lipidated membrane targeting motif of H-ras (tH) and a large bilayer made up of di16∶0-PC (DPPC), di18∶2-PC (DLiPC), and cholesterol. We found that 4–10 tH molecules assemble into clusters that undergo molecular exchange in the sub-μs to μs time scale, depending on the simulation temperature and hence the stability of lipid domains. Driven by the opposite preference of tH palmitoyls and farnesyl for ordered and disordered membrane domains, clustered tH molecules segregate to the boundary of lipid domains. Additionally, a systematic analysis of depalmitoylated and defarnesylated tH variants allowed us to decipher the role of individual lipid modifications in domain-specific nanocluster localization and thereby explain why homologous Ras isoforms form nonoverlapping nanoclusters. Moreover, the localization of tH nanoclusters at domain boundaries resulted in a significantly lower line tension and increased membrane curvature. Taken together, these results provide a unique mechanistic insight into how protein assembly promoted by lipid-modification modulates bilayer shape to generate functional signaling platforms.
To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the Cα atoms of G60 at switch 2 and G10 at the P-loop and the N-Cα-C-O dihedral (ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition.
The responsiveness of cells to external signals, or cell signalling, involves highly regulated sequences of biochemical reactions at the plasma membrane. When receptor proteins in the outer surface of the plasma membrane are activated by environmental signals, they undergo shape change or chemical modification to assemble internal proteins into an organised complex on the inner surface. Specific protein domains and lipids (fats) that are physically attached to proteins tether key components of this complex to the lipid bilayer of the membrane. Scientists have developed cell-based and chemical techniques that allow them to introduce specific membrane-targeting motifs into proteins to produce and study how thismay alter cell behaviour. As a result, a great deal has been learned about the molecular basis of membrane targeting by lipid-modified proteins. This knowledge holds a promise for the rational design of new compounds to prevent defective membrane targeting that could lead to abnormal signalling and the development of diseases.
Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs.
The relaxed complex scheme, a virtual-screening methodology that accounts for protein receptor flexibility, was used to identify a low-micromolar, non-bisphosphonate inhibitor of farnesyl diphosphate synthase. Serendipitously, we also found that several predicted farnesyl diphosphate synthase inhibitors were low-micromolar inhibitors of undecaprenyl diphosphate synthase. These results are of interest because farnesyl diphosphate synthase inhibitors are being pursued as both anti-infective and anticancer agents, and undecaprenyl diphosphate synthase inhibitors are antibacterial drug leads.
Guanine and adenine nucleotide triphosphatases, such as Ras proteins and protein kinases, undergo large conformational changes upon ligand binding in the course of their functions. New computer simulation methods have combined with experimental studies to deepen our understanding of these phenomena. In particular, a 'conformational selection' picture is emerging, where alterations in the relative populations of pre-existing conformations can best describe the conformational switching activity of these important proteins.
A continuum electromechanical model is proposed to describe the membrane curvature induced by electrostatic interactions in a solvated protein-membrane system. The model couples the macroscopic strain energy of membrane and the electrostatic solvation energy of the system, and equilibrium membrane deformation is obtained by minimizing the electroelastic energy functional with respect to the dielectric interface. The model is illustrated with the systems with increasing geometry complexity and captures the sensitivity of membrane curvature to the permanent and mobile charge distributions.
The Kras protein, a member of the Ras family of bio-switches that are frequently mutated in cancer and developmental disorders, becomes functional when anchored to the inner surface of the plasma membrane. It is well known that membrane attachment involves the farnesylated and poylcationic C-terminus of the protein. However, little is known about the structure of the complex and the specific protein-lipid interactions that are responsible for the binding. On the basis of data from extensive (>0.55 μs) molecular dynamics simulations of multiple Kras anchors in bilayers of POPC/POPG lipids (4:1 ratio), we show that, as expected, Kras is tethered to the bilayer surface by specific lysine-POPG salt bridges and by nonspecific farnesyl-phospholipid van der Waals interactions. Unexpectedly, however, only the C-terminal five of the eight Kras Lys side chains were found to directly interact with the bilayer, with the N-terminal ones staying in water. Furthermore, the positively charged Kras anchors pull the negatively charged POPG lipids together, leading to the clustering of the POPG lipids around the proteins. This selective Kras-POPG interaction is directly related to the specific geometry of the backbone, which exists in two major conformational states: 1), a stable native-like ensemble of structures characterized by an extended geometry with a pseudohelical turn; and 2), less stable nonnative ensembles of conformers characterized by severely bent geometries. Finally, although the interface-bound anchor has little effect on the overall structure of the bilayer, it induces local thinning within a persistence length of ∼12 Å. Our results thus go beyond documenting how Kras attaches to a mixed bilayer of charged and neutral lipids; they highlight a fascinating process of protein-induced lipid sorting coupled with the (re)shaping of a surface-bound protein by the host lipids.
The induced fit model has traditionally been invoked to describe the activating conformational change of the monomeric G-proteins, such as Ras and Rho. With this scheme, the presence or absence of the γ-phosphate of GTP leads to an instantaneous switch in conformation. Here we describe atomistic molecular simulations that demonstrate that both Ras and Rho superfamily members harbor an intrinsic susceptibility to sample multiple conformational states in the absence of nucleotide ligand. By comparing the distribution of conformers in the presence and absence of nucleotide, we show that conformational selection is the dominant mechanism by which Ras and Rho undergo nucleotide-dependent conformational changes. Furthermore, the pattern of correlated motions revealed by these simulations predicts a preserved allosteric coupling of the nucleotide-binding site with the membrane interacting C-terminus in both Rho and Ras.
Ras GTPases are membrane-anchored molecular switches that mediate signaling pathways controlling a variety of cellular processes, including cell division and development. Despite their prominent role in many forms of cancer, little is known about the structure of the membrane bound protein or the mechanism and thermodynamics of membrane insertion. The modulation of membrane binding by the catalytic domain is another area of on-going scrutiny. Recent computational and experimental efforts that have begun to shed some light on these issues are the subject of this review. The bulk of the available structural and thermodynamic information on membrane-bound Ras has been obtained by studying peptides derived from the membrane-anchoring regions of N-ras and H-ras proteins. However, those results have been complemented by data, though limited, on the membrane binding of the full-length Ras as well as by predictions about putative communication routes between the GTP-hydrolyzing catalytic domain and the membrane-interacting C-terminus. A tentative mechanistic picture of Ras signaling that is emerging from these studies will be discussed in connection with allostery and implication for the design of selective anti-cancer drugs.
Molecular dynamics (MD) simulation is a popular technique to study bilayer structural properties, but it has not been widely used in mixed bilayers of neutral and charged lipids. Here, we present results from constant temperature and pressure MD simulations of a 2-oleoyl-1-pamlitoyl-sn-glyecro-3-phosphocholine (POPC) bilayer containing 23% 2-oleoyl-1-pamlitoyl-sn-glyecro-3-glycerol (POPG). The simulations were performed using the recently updated CHARMM force field and involved two bilayers of 104 and 416 lipids. A control simulation of a pure POPC bilayer of 128 lipids yielded equilibrium structural properties that compare very well with experimental data. The average equilibrium properties of the mixed bilayer systems were very similar to those of the pure POPC. However, nearly one-half of all the POPG lipids were found to be involved in hydrogen bonding with POPC lipids. Furthermore, the hydration of the mixed bilayer is different from that of the pure POPC, with the former inducing ordering of water molecules at longer distances. Thus, a phospholipid bilayer with ∼23% negative charge content in the liquid crystalline phase differs from its neutral counterpart only at the headgroup.
The precise role of the sphingosine base trans double bond for the unique properties of sphingomyelins (SMs), one of the main lipid components in raftlike structures of biological membranes, has not been fully explored. Several reports comparing the hydration, lipid packing, and hydrogen-bonding behaviors of SM and glycerophospholipid bilayers found remarkable differences overall. However, the atomic interactions linking the double-bond geometry with these thermodynamic and structural changes remained elusive. A recent report on ceramides, which differ from SMs only by their hydroxyl headgroup, has shown that replacing the trans double bond of the sphingosine base by cis weakens the hydrogen-bonding potential of these lipids and thereby alters their biological activity. Based on data from extensive (a total 0.75 μs) atomistic molecular dynamics simulations of bilayers composed of all-trans, all-cis, and a trans/cis (4:1 ratio) racemic mixture of sphingomyelin lipids, here we show that the trans configuration allows for the formation of significantly more hydrogen bonds than the cis. The extra hydrogen bonds enabled tighter packing of lipids in the all-trans and trans/cis bilayers, thus reducing the average area per lipid while increasing the chain order and the bilayer thickness. Moreover, fewer water molecules access the lipid-water interface of the all-trans bilayer than of the all-cis bilayer. These results provide the atomic basis for the importance of the natural sphingomyelin trans double-bond conformation for the formation of ordered membrane domains.
Ras proteins regulate signaling cascades crucial for cell proliferation and differentiation by switching between GTP- and GDP-bound conformations. Distinct Ras isoforms have unique physiological functions with individual isoforms associated with different cancers and developmental diseases. Given the small structural differences among isoforms and mutants, it is currently unclear how these functional differences and aberrant properties arise. Here we investigate whether the subtle differences among isoforms and mutants are associated with detectable dynamical differences. Extensive molecular dynamics simulations reveal that wild-type K-Ras and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. The crucial switch 1 and switch 2 regions along with loop 3, helix 3, and loop 7 contribute to this enhanced flexibility. Removing the gamma-phosphate of the bound GTP from the structure of A59G led to a spontaneous GTP-to-GDP conformational transition in a 20-ns unbiased simulation. The switch 1 and 2 regions exhibit enhanced flexibility and correlated motion when compared to non-transitioning wild-type H-Ras over a similar timeframe. Correlated motions between loop 3 and helix 5 of wild-type H-Ras are absent in the mutant A59G reflecting the enhanced dynamics of the loop 3 region. Taken together with earlier findings, these results suggest the existence of a lower energetic barrier between GTP and GDP states of the mutant. Molecular dynamics simulations combined with principal component analysis of available Ras crystallographic structures can be used to discriminate ligand- and sequence-based dynamic perturbations with potential functional implications. Furthermore, the identification of specific conformations associated with distinct Ras isoforms and mutants provides useful information for efforts that attempt to selectively interfere with the aberrant functions of these species.
Bile acids are powerful detergents that emulsify and solubilize lipids, vitamins, cholesterol and other molecules in the biliary tract and intestines. It has long been known that bile acids form soluble mixed micelles with lipids. However, the detailed thermodynamic and structural properties of these micelles are not fully understood. This study sheds light on this issue based on results from multiple molecular dynamics simulations of cholic acid (CA) and dodecylphosphocholine (DPC) mixed micelles. We found that CA molecules form aggregates of up to 12 monomers with a mean size of 5−6. In agreement with several previous simulations and earlier predictions, the overall shape of these CA clusters is oblate disk-like such that the methyl groups point toward the core of the aggregate and the hydroxyl groups point away from it. The self-aggregation behavior of the CA clusters in the DPC−CA mixture is similar to the pure CA. Furthermore, the sizes and aggregation numbers of the DPC−CA mixed micelles are linearly dependent on CA molarity. In agreement with the radial shell model of Nichols and Ozarowski [Nichols, J. W.; Ozarowski, J. Biochemistry 1990, 29, 4600], our results demonstrate that CA molecules form a wedge between the DPC molecules with their hydroxyl and carboxyl groups facing the aqueous phase while their methyl groups are buried in and face the hydrocarbon core of the DPC micelle. The DPC−CA micelles simulated here tend to be spherical to prolate in shape, with the deviation from spherical geometry significantly increasing with increasing CA:DPC ratio.
The structural elements encoding functional diversity among Ras GTPases are poorly defined. The orientation of the G domain of H-ras with respect to the plane of the plasma membrane is recognized by the Ras binding domain of C-Raf, coupling orientation to MAPK activation. We now show that two other proteins, phosphoinositide- 3-kinase-α and the structurally unrelated galectin-1, also recognize G-domain orientation. These results rationalize the role of galectin-1 in generating active GTP-H-ras signaling nanoclusters. However, molecular dynamics simulations of K-ras membrane insertion and fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) imaging of the effector interactions of NRas, K-Ras, and M-ras suggest that there are two hyperactive, signaling-competent orientations of the Ras G domain. Mutational and functional analyses establish a clear relationship between effector binding and the amphilicities of helix α4 and the C-terminal hypervariable region, thus confirming that these structural elements critically tune the orientation of the Ras G domain. Finally, we show that G-domain orientation and nanoclustering synergize to generate Ras isoform specificity with respect to effector interactions.
An analysis of group IVA (GIVA) phospholipase A(2) (PLA(2)) inhibitor binding was conducted using a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models of the GIVA PLA(2) inhibitors pyrrophenone and the 2-oxoamide AX007 docked into the protein were designed on the basis of deuterium exchange results, and extensive molecular dynamics simulations were run to determine protein-inhibitor contacts. The models show that both inhibitors interact with key residues that also exhibit changes in deuterium exchange upon inhibitor binding. Pyrrophenone is bound to the protein through numerous hydrophobic residues located distal from the active site, while the oxoamide is bound mainly through contacts near the active site. We also show differences in protein dynamics around the active site between the two inhibitor-bound complexes. This combination of computational and experimental methods is useful in defining more accurate inhibitor binding sites and can be used in the generation of better inhibitors against GIVA PLA(2).
Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.
Understanding the mechanism of ion permeation across lipid bilayers is key to controlling osmotic pressure and developing new ways of delivering charged, drug-like molecules inside cells. Recent reports suggest ion-pairing as the mechanism to lower the free energy barrier for the ion permeation in disagreement with predictions from the simple electrostatic models. In this paper we quantify the effect of ion-pairing or charge quenching on the permeation of Na(+) and Cl(-) ions across DMPC lipid bilayer by computing the corresponding potentials of mean force (PMFs) using fully atomistic molecular dynamics simulations. We find that the free energy barrier to permeation reduces in the order Na(+)-Cl(-) ion-pair (27.6 kcal/mol) > Cl(-) (23.6 kcal/mol) > Na(+) (21.9 kcal/mol). Furthermore, with the help of these PMFs we derive the change in the binding free energy between the Na(+) and Cl(-) with respect to that in water as a function of the bilayer permeation depth. Despite the fact that the bilayer boosts the Na(+)-Cl(-) ion binding free energy by as high as 17.9 kcal/mol near its center, ion-pairing between such hydrophilic ions as Na(+) and Cl(-) does not assist their permeation. However, based on a simple thermodynamic cycle, we suggest that ion-pairing between ions of opposite charge and solvent philicity could enhance ion permeation. Comparison of the computed permeation barriers for Na(+) and Cl(-) ions with available experimental data supports this notion. This work establishes general computational methodology to address ion-pairing in fluid anisotropic media and details the ion permeation mechanism on atomic level.
Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25–40 and 57–75) intermediate between GTP and GDP states, or distinct loop3 (46–49), loop7 (105–110), and α5 C-terminus (159–166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of α2 (residues 66–74) with α3-loop7 (93–110), loop2 (26–37) with loop10 (145–151), and loop3 (46–49) with α5 (152–167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.
The functionally required membrane attachment of Ras is achieved through an invariant isoprenylation of a C-terminal Cys, supplemented by further lipid modification of adjacent Cys residues by one (N-ras) or two (H-ras) palmitoyls. However, whether the triply lipidated membrane anchor of H-ras has a higher membrane affinity than its doubly lipidated counterpart, or whether the affinity contribution of the two palmitates and the farnesyl is additive, was not known. To address this issue, we carried out potential of mean force (PMF or free energy profile) calculations on a hexadecylated but nonpalmitoylated anchor (Cys186-HD), hexadecylated and monopalmitoylated anchors (Cys181-monopalmitate and Cys184-monopalmitate), and a nonlipid-modified anchor. We found that the overall insertion free energy follows the trend Cys181/Cys184-bipalmitate (wild type) approximately Cys181-monopalmitate > Cys184-monopalmitate >> nonpalmitoylated anchor. Consistent with suggestions from recent cell biological experiments, the computed PMFs, coupled with structural analysis, demonstrate that membrane affinity of the Ras anchor depends on both the hydrophobicity of the palmitate and the prenyl groups and the spacing between them. The data further suggest that while Cys181-palmitate and Cys186-farnesyl together provide sufficient hydrophobic force for tight membrane binding, the palmitoyl at Cys184 is likely designed to serve another, presumably functional, role.
Understanding the signalling function of Ras GTPases has been the focus of much research for over 20 years. Both the catalytic domain and the membrane anchoring C terminal hypervariable region (HVR) of Ras are necessary for its cellular function. However, while the highly conserved catalytic domain has been characterized in atomic detail, the structure of the full-length membrane-bound Ras has remained elusive. Lack of structural knowledge on the full-length protein limited our understanding of Ras signalling. For example, structures of the Ras catalytic domain solved in complex with effectors do not provide a basis for the functional specificity of different Ras isoforms. Recent molecular dynamics simulations in combination with biophysical and cell biological experiments have shown that the HVR and parts of the G domain cofunction with the lipid tails to anchor H-ras to the plasma membrane. In the GTP-bound state, H-ras adopts an orientation that allows read out by Ras effectors and translation into corresponding MAPK signalling. Here we discuss details of an analysis that suggests a novel balance model for Ras functioning. The balance model rationalizes Ras membrane orientation and may help explain isoform specific interactions of Ras with its effectors and modulators.
To shed light on the driving force for the hydrophobic effect that partitions amphiphilic lipoproteins between water and membrane, we carried out an atomically detailed thermodynamic analysis of a triply lipid modified H-ras heptapeptide anchor (ANCH) in water and in a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Combining molecular mechanical and continuum solvent approaches with an improved technique for solute entropy calculation, we obtained an overall transfer free energy of approximately -13 kcal mol(-1). This value is in qualitative agreement with free energy changes derived from a potential of mean force calculation and indirect experimental observations. Changes in free energies of solvation and ANCH conformational reorganization are unfavorable, whereas ANCH-DMPC interactions-especially van der Waals-favor insertion. These results are consistent with an enthalpy-driven hydrophobic effect, in accord with earlier calorimetric data on the membrane partition of other amphiphiles. Furthermore, structural and entropic analysis of molecular dynamics-generated ensembles suggests that conformational selection may play a hitherto unappreciated role in membrane insertion of lipid-modified peptides and proteins.
A variety of biomolecules mediate physiological processes by inserting and reorganizing in cell membranes, and the thermodynamic forces responsible for their partitioning are of great interest. Recent experiments provided valuable data on the free energy changes associated with the transfer of individual amino acids from water to membrane. However, a complete picture of the pathways and the associated changes in energy of peptide insertion into a membrane remains elusive. To this end, computational techniques supplement the experimental data with atomic-level details and shed light on the energetics of insertion. Here, we employed the technique of umbrella sampling in a total 850 ns of all-atom molecular dynamics simulations to study the free energy profile and the pathway of insertion of a model hexapeptide consisting of a tryptophan and five leucines (WL5). The computed free energy profile of the peptide as it travels from bulk solvent through the membrane core exhibits two minima: a local minimum at the water-membrane interface or the headgroup region and a global minimum at the hydrophobic-hydrophilic interface close to the lipid glycerol region. A rather small barrier of roughly 1 kcal mol (-1) exists at the membrane core, which is explained by the enhanced flexibility of the peptide when deeply inserted. Combining our results with those in the literature, we present a thermodynamic model for peptide insertion and aggregation which involves peptide aggregation upon contact with the membrane at the solvent-lipid headgroup interface.
Ras GTPases are conformational switches controlling cell proliferation, differentiation, and development. Despite their prominent role in many forms of cancer, the mechanism of conformational transition between inactive GDP-bound and active GTP-bound states remains unclear. Here we describe a detailed analysis of available experimental structures and molecular dynamics simulations to quantitatively assess the structural and dynamical features of active and inactive states and their interconversion. We demonstrate that GTP-bound and nucleotide-free G12V H-ras sample a wide region of conformational space, and show that the inactive-to-active transition is a multiphase process defined by the relative rearrangement of the two switches and the orientation of Tyr32. We also modeled and simulated N- and K-ras proteins and found that K-ras is more flexible than N- and H-ras. We identified a number of isoform-specific, long-range side chain interactions that define unique pathways of communication between the nucleotide binding site and the C terminus.
The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and medium-throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix alpha4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP- or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP- or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the beta2-beta3 loop and helix alpha5 are involved in a novel conformational switch that operates through helix alpha4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.
Acetylcholinesterase rapidly hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses, including the neuromuscular junction. The tetramer is the most important functional form of the enzyme. Two low-resolution crystal structures have been solved. One is compact with two of its four peripheral anionic sites (PAS) sterically blocked by complementary subunits. The other is a loose tetramer with all four subunits accessible to solvent. These structures lacked the C-terminal amphipathic t-peptide (WAT domain) that interacts with the proline-rich attachment domain (PRAD). A complete tetramer model (AChEt) was built based on the structure of the PRAD/WAT complex and the compact tetramer. Normal mode analysis suggested that AChEt could exist in several conformations with subunits fluctuating relative to one another. Here, a multiscale simulation involving all-atom molecular dynamics and Cα-based coarse-grained Brownian dynamics simulations was carried out to investigate the large-scale intersubunit dynamics in AChEt. We sampled the ns-μs timescale motions and found that the tetramer indeed constitutes a dynamic assembly of monomers. The intersubunit fluctuation is correlated with the occlusion of the PAS. Such motions of the subunits "gate" ligand-protein association. The gates are open more than 80% of the time on average, which suggests a small reduction in ligand-protein binding. Despite the limitations in the starting model and approximations inherent in coarse graining, these results are consistent with experiments which suggest that binding of a substrate to the PAS is only somewhat hindered by the association of the subunits.
H-, N- and K-ras4B are lipid-anchored, peripheral membrane guanine nucleotide binding proteins. Recent work has shown that Ras proteins are laterally segregated into non-overlapping, dynamic domains of the plasma membrane called nanoclusters. This lateral segregation is important to specify Ras interactions with membrane-associated proteins, effectors and scaffolding proteins and is critical for Ras signal transduction. Here we review biological, in vitro and structural data that provide insight into the molecular basis of how palmitoylated Ras proteins are anchored to the plasma membrane. We explore possible mechanisms for how the interactions of H-ras with a lipid bilayer may drive nanocluster formation.
Ras GTPases become functionally active when anchored to membranes by inserting their lipid modified side chains. Their role in cell division, development, and cancer has made them targets of extensive research efforts, yet the mechanism of membrane insertion and the structure of the resulting complex remain elusive. Recently, the structure of the full-length H-ras protein in a DMPC bilayer has been computationally characterized. Here, the atomic interactions between the H-ras membrane anchor and the DMPC bilayer are investigated in detail. We find that the palmitoylated cysteines and Met182 have dual contributions to membrane affinity: hydrogen bonding by their amides and van der Waals interaction by their hydrophobic side chains. The polar side chains help maintain the orientation of the anchor. Although the overall structure of the bilayer is similar to that of a pure DMPC, there are localized perturbations. These perturbations depend on the insertion depth and backbone localization of the anchor, which in turn is modulated by the catalytic domain and the linker. The pattern of anchor amide-DMPC phosphate/carbonyl hydrogen bonds and the flexibility of Palm184 are important in discriminating between different modes of ras-DMPC interactions. The results provide structural arguments in support of the proposed participation of ras in the organization of membrane nanoclusters.
Ras GTPases mediate signaling pathways in cell proliferation, development and apoptosis. They undergo isoprenylation at a C-terminal CaaX signal (a usually represents aliphatic and X any amino acid) followed by proteolysis of aaX and carboxymethylation. In the case of H-ras, a subsequent dual palmitoylation of cysteines adjacent to the site of farnesylation produces a mature anchor for plasma membrane targeting.[1,2] Atomistic informations, such as the structure of membrane-bound ras and the free energy of complex formation, are vital in research efforts geared towards designing ras isoform selective anti-cancer agents. The most common experimental techniques are not yet able to provide such atomistic data. Here we present computational results on the free energy profile for the transfer of the H-ras membrane anchor from water to a bilayer of DMPC lipids. We find that there is no significant barrier for insertion and that once a few carbon atoms of the ras lipid chains cross the membrane-water interface, the free energy displays a steeply downhill profile. Insertion into the hydrocarbon core of the ras lipids and the interfacial localization of the backbone together produce up to 30 kcal/mol gain in free energy. Additionally, using the recently reported computationally-derived structures of full-length H-ras in a DMPC bilayer,[3] we explain how a small free energy difference would enable modulation of H-ras membrane binding by the linker and the catalytic domain.
The self-perpetuating conversion of cellular prion proteins (PrP(C)) into an aggregated beta-sheet rich conformation is associated with transmissible spongiform encephalopathies (TSE). The loop 166-175 (L1) in PrP(C), which displays sequence and structural variation among species, has been suggested to play a role in species barrier, in particular against transmission of TSE from cervids to domestic and laboratory animals. L1 is ordered in elk PrP, as well as in a mouse/elk hybrid (in which L1 of mouse is replaced by elk) but not in other species such as mice, humans, and bovine. To investigate the source and significance of L1 dynamics, we carried out explicit solvent molecular dynamics simulations (approximately 0.5 micros in total) of the mouse prion protein, the mouse/elk hybrid, and control simulations, in which the mouse sequence is reintroduced into the structure of the mouse/elk hybrid. We found that the flexibility of L1 correlates with the backbone dynamics of Ser170. Furthermore, L1 mobility promotes a substantial displacement of Tyr169, rupture of the Asp178-Tyr128 and Asp178-Tyr169 side chain hydrogen bonds, as well as disruption of Tyr169-Phe175 pi-stacking interaction. The simulation results go beyond the available experimental data because they highlight the dependence of this network of interactions on residue 170 and L1 plasticity.
Peptide insertion, positioning, and stabilization in a model membrane are probed via an all-atom molecular dynamics (MD) simulation. One peptide (WL5) is simulated in each leaflet of a solvated dimyristoylglycero-3-phosphate (DMPC) membrane. Within the first 5 ns, the peptides spontaneously insert into the membrane and then stabilize during the remaining 70 ns of simulation time. In both leaflets, the peptides localize to the membrane interface, and this localization is attributed to the formation of peptide-lipid hydrogen bonds. We show that the single tryptophan residue in each peptide contributes significantly to these hydrogen bonds; specifically, the nitrogen heteroatom of the indole ring plays a critical role. The tilt angles of the indole rings relative to the membrane normal in the upper and lower leaflets are approximately 26 degrees and 54 degrees , respectively. The tilt angles of the entire peptide chain are 62 degrees and 74 degrees . The membrane induces conformations of the peptide that are characteristic of beta-sheets, and the peptide enhances the lipid ordering in the membrane. Finally, the diffusion rate of the peptides in the membrane plane is calculated (based on experimental peptide concentrations) to be approximately 6 A(2)/ns, thus suggesting a 500 ns time scale for intermolecular interactions.
Ras proteins regulate signal transduction processes that control cell growth and proliferation. Their disregulation is a common cause of human tumors. Atomic level structural and dynamical information in a membrane environment is crucial for understanding signaling specificity among Ras isoforms and for the design of selective anti-cancer agents. Here, the structure of the full-length H-Ras protein in complex with a 1,2-dimyristoylglycero-3-phosphocholine (DMPC) bilayer obtained from modeling and all-atom explicit solvent molecular dynamics simulations, as well as experimental validation of the main results, are presented. We find that, in addition to the lipid anchor, H-Ras membrane binding involves direct interaction of residues in the catalytic domain with DMPC phosphates. Two modes of binding (possibly modulated by GTP/GDP exchange) differing in the orientation and bilayer contact of the soluble domain as well as in the participation of the flexible linker in membrane binding are proposed. These results are supported by our initial in vivo experiments. The overall structures of the protein and the bilayer remain similar to those of the isolated components, with few localized structural and dynamical changes. The implications of the results to membrane lateral segregation and other aspects of Ras signaling are discussed.
The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.
Lipid-modified membrane-binding proteins are essential in signal transduction events of the cell, a typical example being the GTPase ras. Recently, membrane binding of a doubly lipid-modified heptapeptide from the C-terminus of the human N-ras protein was studied by spectroscopic techniques. It was found that membrane binding is mainly due to lipid chain insertion, but it is also favored by interactions between apolar side chains and the hydrophobic region of the membrane. Here, 10 explicit solvent molecular dynamics simulations for a total time of about 150 ns are used to investigate the atomic details of the peptide-membrane association. The 16:0 peptide lipid chains are more mobile than the 14:0 phospholipid chains, which is in agreement with (2)H NMR experiments. Peptide-lipid and peptide-solvent interactions, backbone and side-chain distributions, as well as the effects of lipidated peptide insertion onto the structure, and dynamics of a 1,2-dimyristoylglycero-3-phosphocholine bilayer are described. The simulation results validate the structural model proposed by the analysis of spectroscopic data and highlight the main aspects of the insertion mechanism. The peptide in the membrane is rather rigid over the simulation time scale of about 10 ns, but different partially extended conformations devoid of backbone hydrogen bonds are observed in different trajectories.
The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.
The N-terminal domain of the bacterial integrase Tn916 specifically recognizes the 11 bp DNA target site by positioning the face of a three-stranded beta-sheet into the major groove. Binding is linked to structural adaptation. We have characterized INT-DBD binding to DNA in detail by calorimetry [Milev, S., Gorfe, A., Karshikoff, A., Clubb, R. T., Bosshard, H. R., and Jelesarov, I. (2003) Biochemistry 42, 3481-3491]. Our thermodynamic analysis has indicated that the major driving force of association is the hydrophobic effect while polar interactions contribute less. To gain more comprehensive information about the binding process, we performed a computational analysis of the binding free energy and report here the results. A hybrid molecular mechanics/continuum approach was followed. The total binding free energy is predicted with reasonable accuracy. The calculations confirm that nonpolar effects stabilize the protein-DNA complex while electrostatics opposes binding. Structural changes optimizing surface complementarity are costly in terms of energy. The energetic consequences from the replacement of nine DNA-contacting residues by alanine were investigated. The calculations correctly predict the binding affinity decrease of eight mutations and the destabilizing effect of one wild-type residue. Bulky side chains stabilize the wild-type complex through packing interactions and favorable nonpolar dehydration, but the net nonpolar energy changes do not correlate with the relative affinity loss upon mutation. Discrete protein-DNA electrostatic interactions may be net stabilizing or net destabilizing depending on the local environment. In contrast to nonpolar energy changes, the magnitude of the electrostatic free energy ranks the mutations according to the experimentally measured DeltaDeltaG. Free energy decomposition analysis from a structural perspective leads to detailed information about the thermodynamic strategy used by INT-DBD for sequence-specific DNA binding.
Sequence-specific DNA recognition by bacterial integrase Tn916 involves structural rearrangements of both the protein and the DNA duplex. Energetic contributions from changes of conformation, thermal motions and soft vibrational modi of the protein, the DNA, and the complex significantly influence the energetic profile of protein-DNA association. Understanding the energetics of such a complicated system requires not only a detailed calorimetric investigation of the association reaction but also of the components in isolation. Here we report on the conformational stability of the integrase Tn916 DNA binding domain and its cognate 13 base pair target DNA duplex. Using a combination of temperature and denaturant induced unfolding experiments, we find that the 74-residue DNA binding domain is compact and unfolds cooperatively with only small deviation from two-state behavior. Scanning calorimetry reveals an increase of the heat capacity of the native protein attributable to increased thermal fluctuations. From the combined calorimetric and spectroscopic experiments, the parameters of protein unfolding are T(m) = 43.8 +/- 0.3 degrees C, DeltaH(m) = 255 +/- 18 kJ mol(-1), DeltaS(m) = 0.80 +/- 0.06 kJ mol(-1), and DeltaC(p) = 5.0 +/- 0.8 kJ K(-1) mol(-1). The DNA target duplex displays a thermodynamic signature typical of short oligonucleotide duplexes: significant heat absorption due to end fraying and twisting precedes cooperative unfolding and dissociation. The parameters for DNA unfolding and dissociation are DeltaH(m) = 335 +/- 4 kJ mol(-1) and DeltaC(p) = 2.7 +/- 0.9 kJ K(-(1) mol(-1). The results reported here have been instrumental in interpreting the thermodynamic features of the association reaction of the integrase with its 13 base pair target DNA duplex reported in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3481-3491].
The DNA binding domain of the transposon Tn916 integrase (INT-DBD) binds to its DNA target site by positioning the face of a three-stranded antiparallel beta-sheet within the major groove. Binding of INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry, differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, and fluorescence spectroscopy. The observed heat capacity change accompanying the association reaction (DeltaC(p)) is temperature-dependent, decreasing from -1.4 kJ K(-1) mol(-1) at 4 degrees C to -2.9 kJ K(-1) mol(-1) at 30 degrees C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and the protein-DNA complex are not changing in parallel when the temperature increases and that thermal motions of the protein and the DNA are restricted in the complex. After correction for this effect, DeltaC(p) is -1.8 kJ K(-1) mol(-1) and temperature-independent. However, this value is still higher than DeltaC(p) of -1.2 kJ K(-1) mol(-1) estimated by semiempirical methods from dehydration of surface area buried at the complex interface. We propose that the discrepancy between the measured and the structure-based prediction of binding energetics is caused by incomplete dehydration of polar groups in the complex. In support, we identify cavities at the interface that are large enough to accommodate approximately 10 water molecules. Our results highlight the difficulties of structure-based prediction of DeltaC(p) (and other thermodynamic parameters) and emphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complex but also of the folding thermodynamics of the protein and the DNA alone, which are described in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].
Interhelical salt bridges are common in leucine zippers and are thought to stabilize the coiled coil conformation. Here we present a detailed thermodynamic investigation of the designed, disulfide-linked leucine zipper AB(SS) whose high-resolution NMR structure shows six interhelical ion pairs between heptad positions g of one helix and e' of the other helix but no ion pairing within single helices. The average pK(a) value of the Glu side chain carboxyl groups of AB(SS) is slightly higher than the pK(a) of a freely accessible Glu in an unfolded peptide [Marti, D. N., Jelesarov, I., and Bosshard, H. R. (2000) Biochemistry 39, 12804-12818]. This indicates that the salt bridges are destabilizing, a prediction we now have confirmed by determining the pH +/- stability profile of AB(SS). Circular dichroism-monitored unfolding by urea and by heating and differential scanning calorimetry show that the coiled coil conformation is approximately 5 kJ/mol more stable when salt bridges are broken by protonation of the carboxyl side chains. Using guanidinium chloride as the denaturant, the increase in the free energy of unfolding on protonation of the carboxyl side chains is larger, approximately 17 kJ/mol. The discrepancy between urea and guanidinium chloride unfolding can be ascribed to the ionic nature of guanidinium chloride, which screens charge-charge interactions. This work demonstrates the difficulty of predicting the energetic contribution of salt bridges from structural data alone even in a case where the ion pairs are seen in high-resolution NMR structures. The reason is that the contribution to stability results from a fine balance between energetically favorable Coulombic attractions and unfavorable desolvation of charges and conformational constraints of the residues involved in ion pairing. The apparent discrepancy between the results presented here and mutational studies indicating stabilization by salt bridges is discussed and resolved. An explanation is proposed for why interhelical salt bridges are frequently found in natural coiled coils despite evidence that they do not directly contribute to stability.
The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pK(a) calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pK(a) estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve-averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pK(a) calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pK(a) predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pK(a) predictions for the model leucine zipper was reduced to as low as approximately 0.25 pK(a) units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pK(a) calculations, are discussed.
A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson-Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S(1) region of all trypsins is generated from Asp(189), but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S(1)-pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S(1)-pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S(1) pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes.


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Dr. Robin Plank specializes in family practice in Slippery Rock, Pennsylvania.
Dr. Robin Plank has been compared 89 times. Details of Dr. Plank's 16 years experience as an DO, her hospital affiliation and education at Philadelphia College of Osteopathic Medicine are available on your free doctor report.
Primary Location for Dr. Plank
100 Innovation Dr
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Slippery Rock, PA 16057
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Dr. Plank attended medical school at Philadelphia College of Osteopathic Medicine and graduated in 1999 having 16 years experience.
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Felice Tilin, Ph.D.
Director, Graduate Organization Development and Leadership
Denver H. Travis Ph.D., CFA
Assistant Professor
William Trombetta, Ph.D.
Stephanie A. Tryce, J.D.
Assistant Professor
John Tudor, Ph.D.
Professor Emeritus
Tesfalidet Tukue
John Urbanchuk, Ph.D.
John Vacca, Ph.D.
Associate Dean, CAS and Director of the College of Liberal and Professional Studies
Captain Nicholas A. Vallera
Assistant Professor, Operations Flight Commander of AFROTC Detachment 750
Tony Van Dyke
Brice R. Wachterhauser, Ph.D.
Aubrey Wang, Ph.D.
Associate Professor, Director of Interdisciplinary Doctor of Education Program for Educational Leaders
Richard A. Warren, Ph.D.
Professor and Interim Dean of the College of Arts & Sciences
James Watrous, Ph.D.
Mareile Watson
George Webster, Ph.D.
Associate Professor Emeritus
Dennis Weeks, M.F.A.
Associate Professor
Susanna Wei, Ph.D.
Associate Professor
Thani Jambulingam, Ph.D.


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by Sam P.K. Collins Mar 6, 2015
Could this be the beginning of a national trend?
by Sam P.K. Collins Mar 3, 2015
Price surges for naloxone could deter efforts to curb heroin overdoses.
by Sam P.K. Collins Feb 26, 2015
A new report outlines the sources of the most prevalent foodborne pathogens.
by Sam P.K. Collins Feb 24, 2015
Even with new infection rates falling, the fight rages on and questions remain.
by Sam P.K. Collins Feb 23, 2015
At least 25 percent of the country's homeless youth need mental health services.
by Sam P.K. Collins Jan 30, 2015
"Tobacco use remains the leading preventable cause of death and it [affects] almost every system in the body."
by Tara Culp-Ressler Jan 7, 2015
If you're among the 60 percent of adults who don't get vaccinated against influenza, listen up.
by Sam P.K. Collins Dec 24, 2014
"[W]e have to think broadly about lots of different ways to make families not be reliant on fast food," the lead researcher says."
by Sam P.K. Collins Dec 10, 2014
Amid a national opioid addiction epidemic, this study shows a changing tide among a group that has historically enjoyed a profitable relationship with pharmaceutical companies.
by Sam P.K. Collins Dec 8, 2014
Despite a growing anti-vaccine movement, concerns about vaccination policies loom large among parents choosing a daycare for their child.


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Cops Arrest a Long Island Woman for Dropping Off a Sick Cat at a Clinic, Cat Puns Abound
A suburban retiree with a heart of gold. A cat with swollen ears. A jealous bagel shop worker. Something cute is bound to happen with this crazy crew.
Or not. Yesterday Nancy Glassman was arrested at her Long Island home for snatching a cat from an Upper East Side bagel shop and whisking it away to an animal clinic. Glassman, seeing the cat at the back of the store had ears appearing "five times too big," decided to get him some medical attention before Bagels & Co. worker Youal Aroety called the NYPD on her.
Glassman, 50, a retired optometrist whose old practice was based in Sunset Park, was in town looking at real estate and also for a martial arts class (Why not?) Popping into the corner bagel store just to use the bathroom, she noticed something was off with Costco. She escaped out the back door with the animal.
The Daily News reports that Glassman was visited at her home in North Woodmere by police officers and brought to the 19th Precinct in the Upper East Side to identify the cat, named Costco.
Once she positively ID'd him, the police took her into custody and charged her with burglary, a felony that carries the potential for up to seven years in prison.
The worst part of this story: The cat puns in the original report. Even the NYPD spokesman consulted by the News was in the groaner-making spirit: "'There's no pussyfooting around it," NYPD spokesman John McCarthy said.
Some other zingers:
"She's a cat burglar who got nabbed in a stop-and-frisky."
"... the cat-astrophe began ..."
"It all seemed purr-fectly routine until she was handcuffed and arrested at the precinct."
The double-worst part of this story: The vet told Glassman that Costco has a congenital deformity and does not have long to live. No lolz for this cat, sad to say.
Send your
story tips cat puns to the author, Raillan Brooks.
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Yes, There Is a McDonald's Secret Menu — Time to Chow Down
The biggest news since the Big Mac became, well, big, is here.
The biggest news since the Big Mac became, well, big, is here.
It's time for defenders of the Confederate flag in South Carolina to face the music.
ABC's upcoming Muppets mockumentary series only had so much to show at its recent Comic-Con 2015 panel, but with an actual premiere as far away as September, Kermit, Ms. Piggy, Gonzo and the gang dug deep for a special preview. Check out the 10-minute presentation that convinced ABC to make a new Muppets series!
You don't need money to have a blast with the kids!
A cameraman with New Zealand's TV3 caught the full force of Phil Rudd's rage when he asked the troubled AC/DC drummer about his most recent run in with authorities.
This soldier wasn't just returning home. He was returning to home plate.
What's the only thing scarier than seeing a bear? How about two bears? That are fighting.
Peter Frampton, Cheap Trick and Gregg Allman were just a few of the artists on hand for Lynyrd Skynyrd's all-star concert recording One More for the Fans.
Comedian Denis Leary, in a new talk with Ultimate Classic Rock, says he understands why bands break up – just not why they don't get back together.
Water slides and summer go together like peanut butter and jelly.
Marvel's Ant-Man opens in theaters today and to this point we've seen no shortage of trailers, clips and TV spots showing off all kinds of footage. But, on the last stop on his promotional tour for the movie, Paul Rudd stopped by to visit Conan O'Brien and brought something extra special.
It's never good to upstage the bride on her wedding day -- especially when you work for her.


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Kernel for Solaris (SPARC)4.01
editor's review
buy now $349.30
There are many people who work on more than one device, in order to increase their productivity, and these devices do not always run the same operating system - namely, one of them could run Windows, another one Mac OS, whereas the third one could run a distribution of Linux, for example Solaris Sparc.
However, if data on your Solaris device has been damaged and you want to retrieve it, you can turn to your Windows computer and rely on Kernel for Solaris (SPARC).
Connect your corrupted Solaris Sparc hard drive to your PC
Once you have installed the application, the first step you need to take is to connect the hard drive where the corrupted files are stored, to your computer.
It will automatically be detected within the main window of the app, as some details are instantly displayed, such as the model, capacity, cylinders or sectors per track. Moreover, you can choose the volume identification option that you prefer, be it partition table or sector range.
Thoroughly scan the HDD and identify its partitions
Next, you need to set Kernel for Solaris (SPARC) to perform an in-depth analysis of the connected hard drive so as to detect its partitions as accurately as possible.
This step might take a long time to complete, depending on the storage size of the HDD, yet it is an important one, as identifying the partitions can help the app to locate the damaged files you want to restore.
Easily browse through recoverable files and select the ones you need
When the aforementioned process is finished, you are presented with a detailed list of files so you can explore and mark the ones you want to recover, without too much hassle.
All in all, the application comes in handy to all those who want to use a Windows computer to restore damaged files from their Solaris Sparc hard drive, while also enjoying a neat and straightforward GUI.
top FREE alternatives
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