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SERCA is an important model system for understanding the molecular details of conformational change in membrane transport systems. This reflects the large number of solved X-ray structures and the equally large database of mutations that have been assayed. In this computational study, we provide a molecular dynamics description of the conformational changes during the E1P → E2P transitions. This set of states further changes with insertion mutants in the A-M3 linker region. These mutants were experimentally shown to lead to significant shifts in rates between the E1P → E2P states. Using the population shift framework and dynamic importance sampling method along with coarse-grained representations of the protein, lipid, and water, we suggest why these changes are found. The calculations sample on intermediates and suggest that changes in interactions, individual helix interactions, and water behavior are key elements in the molecular compositions that underlie shifts in kinetics. In particular, as the insertion length grows, it attracts more water and disrupts domain interactions, creating changes as well at the sites of key helix interactions between the A-Domain and the P-Domain. This provides a conceptual picture that aids understanding of the experimental results.

How a biological system responds to a charge shift is a challenging question directly relevant to biological function. Time-resolved fluorescence of a tryptophan residue reflects protein and solvent response to the difference in pi-electron density between the excited and the ground state. In this study we use molecular dynamics to calculate the time-dependent spectral shift (TDSS) in the fluorescence of Trp-43 in GB1 protein. A new computational method for separating solvent, protein, and fluorophore contributions to TDSS is applied to 100 nonequilibrium trajectories for GB1 in TIP3P water. The results support several nontrivial conclusions. Both longitudinal and transverse relaxation modes of bulk solvent contribute to the TDSS in proteins. All relaxation components slower than the transverse relaxation of bulk solvent have significant contributions from both protein and solvent, with a negative correlation between them. Five exponential terms in the TDSS of GB1 are well separated by their relaxation times. A 0.036 ps term is due to both solvent (60%) and protein (40%). Two exponential terms represent longitudinal (tau(L) approximately = 0.4 ps) and transverse (tau(D) approximately = 5.6 ps) relaxation modes of TIP3P water. A 131 ps term is attributable to a small change in the tertiary structure, with the alpha-helix moving 0.2 A away from the beta-strand containing Trp-43. A 2580 ps term is due to the change in the conformation of the Glu-42 side chain that brings its carboxyl group close to the positively charged end of the excited fluorophore. Interestingly, water cancels 60% of the TDSS resulting from this conformational change.

The fusion between two lipid bilayers involves crossing a complicated energy landscape. The limiting barrier in the process appears to be between two closely opposed bilayers, and the intermediate state where the outer leaflets are fused. We have performed molecular dynamics simulations to characterize the free energy barrier for the fusion of two liposomes and to examine the molecular details of barrier crossing. In order to capture the slow dynamics of fusion, a model using coarse-grained representations of lipids was used. The fusion between pairs of liposomes was simulated for four systems: DPPC, DOPC, a 3:1 mixture of DPPC/DPPE and an asymmetric lipid tail system in which one tail of DPPC was reduced to half the length (ASTail). The weighted histogram method was used to compute the free energy as a function of separation distance. The relative barrier heights for these systems was found to be ASTail≫DPPC>DPPC/DPPE>DOPC, in agreement with experimental observations. Further, the free energy curves for all four can be overlayed on a single curve by plotting the free energy versus the surface separation (differing only in the point of fusion). These simulations also confirm that the two main contributions to the free energy barrier are the removal of water between the vesicles and the deformation of the vesicle. The most prominent molecular detail of barrier crossing in all cases examined was the splaying of lipid tails, where a single splayed lipid formed a bridge between the two outer leaflets that promotes additional lipid mixing between the vesicles and eventually leads to fusion. The tail splay appears to be closely connected to the energetics of the process. For example, the high barrier for the ASTail is the result of a smaller distance between terminal methyl groups in the splayed molecule. The shortening of this distance requires the liposomes to be closer together, which significantly increases the cost of water removal and bilayer deformation. Before tail splay can initiate fusion, contact must occur between a tail end and the external water. In isolated vesicles, the contact fraction is correlated to the fusogenicity difference between DPPC and DOPC. Moreover, for planar bilayers the contact fraction is much lower for DPPC, which is consistent with its lack of fusion in giant vesicles. The simulation results show the key roles of lipid tail dynamics in governing the fusion energy landscape.

Using 237 all-atom double bilayer simulations, we examined the thermodynamic and structural changes that occur as a phosphatidylcholine lipid bilayer stack is dehydrated. The simulated system represents a micropatch of lipid multilayer systems that are studied experimentally using surface force apparatus, atomic force microscopy and osmotic pressure studies. In these experiments, the hydration level of the system is varied, changing the separation between the bilayers, in order to understand the forces that the bilayers feel as they are brought together. These studies have found a curious, strongly repulsive force when the bilayers are very close to each other, which has been termed the "hydration force," though the origins of this force are not clearly understood. We computationally reproduce this repulsive, relatively free energy change as bilayers come together and make qualitative conclusions as to the enthalpic and entropic origins of the free energy change. This analysis is supported by data showing structural changes in the waters, lipids and salts that have also been seen in experimental work. Increases in solvent ordering as the bilayers are dehydrated are found to be essential in causing the repulsion as the bilayers come together.

Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis–trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system.

The human erythrocyte sialoglycoprotein glycophorin A (GpA) has been used extensively in experiment and simulations as a model of transmembrane helix-dimer formation, emphasizing the critical role of specific residue-residue interactions between helices in dimer stability. While the tertiary dimer structure is modulated by the hydrophobic lipid bilayer environment, we show that interactions of GpA with ordered interfacial water are commensurate to intrahelical forces. The role of lipid-water interface in stabilizing transmembrane proteins is not yet understood; however, dramatic water reordering in the presence of the transmembrane domains is observed from simulations and is possibly measurable by experiment. Interfacial interactions including anisotropic interactions with the polar headgroups might favor parallel association of transmembrane helices. To quantify forces capable of disrupting the GpA dimer, we generate folding/unfolding intermediates by replacement of the lipid bilayer with water, eliminating not only the native hydrophobic environment but also the native interfacial water region. Dramatic changes in the secondary, helical structures occur, with a transition from i,i+4 α-helix to i,i+5 π-helix and concomitant perturbation of the tertiary structure. Enforcing the native α-helix secondary structure by soft dihedral restraints restores the native tertiary structure, in essence substituting for the lack of the lipid-water interface. We suggest that differentiation between interactions within the lipid bilayer, including interactions with lipid headgroups and interfacial water can enrich our understanding of the thermodynamic stability of transmembrane domains.

The association between monovalent salts and neutral lipid bilayers is known to influence global bilayer structural properties such as headgroup conformational fluctuations and the dipole potential. The local influence of the ions, however, has been unknown due to limited structural resolution of experimental methods. Molecular dynamics simulations are used here to elucidate local structural rearrangements upon association of a series of monovalent Na+ salts to a palmitoyl-oleoyl-phosphatidylcholine bilayer. We observe association of all ion types in the interfacial region. Larger anions, which are meant to rationalize data regarding a Hofmeister series of anions, bind more deeply within the bilayer than either Cl− or Na+. Although the simulations are able to reproduce experimentally measured quantities, the analysis is focused on local properties currently invisible to experiments, which may be critical to biological systems. As such, for all ion types, including Cl−, we show local ion-induced perturbations to headgroup tilt, the extent and direction of which is sensitive to ion charge and size. Additionally, we report salt-induced ordering of the water well beyond the interfacial region, which may be significant in terms of hydration repulsion between stacked bilayers.

The light-driven photocycle of rhodopsin begins the photoreceptor cascade that underlies visual response. In a sequence of events, the retinal covalently attached to the rhodopsin protein undergoes a conformational change that communicates local changes to a global conformational change throughout the whole protein. In turn, the large-scale protein change then activates G-proteins and signal amplification throughout the cell. The nature of this change, involving a coupling between a local process and larger changes throughout the protein, may be important for many membrane proteins. In addition, functional work has shown that this coupling occurs with different efficiency in different lipid settings. To begin to understand the nature of the efficiency of this coupling in different lipid settings, we present a molecular dynamics study of rhodopsin in an explicit dioleoyl-phosphatidylcholine bilayer. Our system was simulated for 40 ns and provides insights into the very early events of the visual cascade, before the full transition and activation have occurred. In particular, we see an event near 10 ns that begins with a change in hydrogen bonding near the retinal and that leads through a series of coupled changes to a shift in helical tilt. This type of event, though rare on the molecular dynamics time-scale, could be an important clue to the types of coupling that occur between local and large-scale conformational change in many membrane proteins.

There is increasing evidence that intrinsically unstructured proteins or protein domains have important biological functions. These types of proteins may be productively analyzed using polymer theory developed to predict global physical properties of polymers. In these theories molecular detail is "coarse grained" out of the models, and replaced with a small number of parameters that characterize the polymer. This reduction in complexity allows extremely large systems to be studied. In the case of simulations, the time scales accessible also increase significantly. Here we discuss the application of polymer theory to unstructured proteins, and consider how to classify proteins within a polymer framework. We then review polymer theory that is relevant to predicting functionally important properties, such as radius of gyration, height of a polymer brush and force required to compress a polymer brush.

The Symposium ‘Frontiers in membrane and membrane protein biophysics: experiments and theory’, held this year at the University of California, Irvine (August 19–20), celebrated the 70th Birthday of Stephen H. White by bringing together distinguished experimentalists and theoreticians to discuss the state of the art and future challenges in the field of membrane and membrane protein biophysics. The meeting and this special issue highlight the highly interdisciplinary nature of membrane and membrane protein biophysics, and the tremendous contributions that S. H. White and his lab have brought to the field.

Molecular dynamics simulations of two structurally similar fatty acid-binding proteins interacting with stearic acid are described. The calculations relate to recent ligand binding measurements and suggest similarities and differences between the two systems. Charged and neutral forms of the fatty acid were examined. The charged forms led to rapid trajectory divergence, whereas the protonated forms remained stable over the length of their 1-ns production trajectories. The two protein systems showed similar sets of total interaction energies with the ligand. However, the strengths of individual amino acids interacting with the ligand differ. Furthermore, covariance analysis of the ligand with both protein and water suggests that the stearic acid in the adipocyte fatty acid-binding protein is coupled more strongly to the water than to the protein. The stearic acid in the muscle fatty acid-binding protein is seen to be coupled differentially along the length of the chain to the protein. These differences could help to rationalize the stronger binding affinity for stearic acid in the human muscle fatty acid-binding protein. An importance scale, based on both covariance and interaction energy with the ligand, is proposed to identify residues that may be important for binding function.

The concepts of hydrophobicity and hydrophobic moments have been applied in attempts to predict membrane protein secondary and tertiary structure. The current paper uses molecular dynamics computer calculations of individual bacteriorhodopsin helices in explicit dimyristoylphosphatidylcholine bilayers to examine the atomic basis of these approaches. The results suggest that the types of interactions between a particular amino acid and the surrounding bilayer depend on the position and type of the amino acid. In particular, aromatic residues are seen to interact favorably at the interface region. Analysis of the trajectories in terms of hydrophobic moments suggests the presence of a particular face that prefers lipid. The results of these simulations may be used to improve secondary structure prediction methods and to provide further insights into the two-stage model of protein folding.

The membrane protein pump SERCA, is a Ca-ATPse that is well-characterized from a structural perspective, with x-ray conformations known for the major states of the pump cycle. Yet, understanding the role of the environment in supporting a state and in enabling transitions between states continues to be a major research challenge. To address these environmental influences we have concentrated our efforts on two of the pump cycle transitions. We now present analysis of a set of four all-atom explicit bilayer simulations of SERCA states (1WPG, 2AGV, 3AR2, and 2ZBD) that we performed using Anton (PSC) and Lonestar (XSEDE) resources. These are to be compared with our previous computations of SERCA using implicit solvent and coarse-grained models. The prior results (J. Mol. Biol.422:575-593 (2012) ; Proteins 80(8):1929-1947 (2012)) suggested an important role of compensation between different environment and protein domains as central to the transitions. We now extend those results with analysis of the explicit bilayer, water, salt and co-factor simulations. An intriguing initial result is that the presence of calcium changed the fluctuations seen in the 3AR2 state.

A molecular dynamics simulation of a simple model membrane system composed of a single amphiphilic helical peptide (ace-K2GL16K2A-amide) in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer was performed for a total of 1060 ps. The secondary structure of the peptide and its stability were described in terms of average dihedral angles, phi and psi, and the C alpha torsion angles formed by backbone atoms; by the average translation per residue along the helix axis; and by the intramolecular peptide hydrogen bonds. The results indicated that residues 6 through 15 remain in a stable right-handed alpha-helical conformation, whereas both termini exhibit substantial fluctuations. A change in the backbone dihedral angles for residues 16 and 17 is accompanied by the loss of two intramolecular hydrogen bonds, leading to a local but long-lived disruption of the helix. The dynamics of the peptide was characterized in terms of local and global helix motions. The local motions of the N-H bond angles were described in terms of the autocorrelation functions of P2[cos thetaNH(t, t + tau)] and reflected the different degrees of local peptide order as well as a variation in time scale for local motions. The chi1 and chi2 dihedral angles of the leucine side chains underwent frequent transitions between potential minima. No connection between the side-chain positions and their mobility was observed, however. In contrast, the lysine side chains displayed little mobility during the simulation. The global peptide motions were characterized by the tilting and bending motions of the helix. Although the peptide was initially aligned parallel to the bilayer normal, during the simulation it was observed to tilt away from the normal, reaching an angle of approximately 25 degrees by the end of the simulation. In addition, a slight bend of the helix was detected. Finally, the solvation of the peptide backbone and side-chain atoms was also investigated.

Understanding the role of the lipid bilayer in membrane protein structure and dynamics is needed for tertiary structure determination methods. However, the molecular details are not well understood. Molecular dynamics computer calculations can provide insight into these molecular details of protein:lipid interactions. This paper reports on 10 simulations of individual alpha-helices in explicit lipid bilayers. The 10 helices were selected from the bacteriorhodopsin structure as representative alpha-helical membrane folding components. The bilayer is constructed of dimyristoyl phosphatidylcholine molecules. The only major difference between simulations is the primary sequence of the alpha-helix. The results show dramatic differences in motional behavior between alpha-helices. For example, helix A has much smaller root-mean-squared deviations than does helix D. This can be understood in terms of the presence of aromatic residues at the interface for helix A that are not present in helix D. Additional motions are possible for the helices that contain proline side chains relative to other amino acids. The results thus provide insight into the types of motion and the average structures possible for helices within the bilayer setting and demonstrate the strength of molecular simulations in providing molecular details that are not directly visualized in experiments.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.

The article by Knecht and Klasczyk in this issue of Biophysical Journal determines similar binding affinities for Na and Cl interacting with POPC membranes (1xSpecific binding of chloride ions to lipid vesicles and implications at molecular scale. Knecht, V. and Klasczyk, B. Biophys. J. 2013; 104: 818–824Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References1). Their joint experimental and computational refinement shows the power of combining data from multiple experimental and computational approaches. Understanding the molecular nature of these interactions is important for membrane protein function, bilayer dynamics, and cellular biophysics. Early work with membrane electrostatics showed the importance of the placement of charge and suggested that dielectric screening could be a functional component of membrane behavior (2xEnergy of an ion crossing a low dielectric membrane: solutions to four relevant electrostatic problems. Parsegian, A. Nature. 1969; 221: 844–846Crossref | PubMed | Scopus (529)See all References, 3xElectrostatic interactions in membranes and proteins. Honig, B.H., Hubbell, W.L., and Flewelling, R.F. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 163–193Crossref | PubMedSee all References). From that beginning point, detailed molecular dynamics simulations have begun to illustrate that the behavior of cations and anions may or may not vary within different regions of the membrane bilayer (4xAqueous solutions at the interface with phospholipid bilayers. Berkowitz, M.L. and Vacha, R. Acc. Chem. Res. 2012; 45: 74–82Crossref | PubMed | Scopus (36)See all References, 5xNaCl interactions with phosphatidylcholine bilayers do not alter membrane structure but induce long-range ordering of ions and water. Valley, C.C., Perlmutter, J.D...., and Sachs, J.N. J. Membr. Biol. 2011; 244: 35–42Crossref | PubMed | Scopus (14)See all References). This, in turn, could lead to significant changes in bilayer shape and function as the concentration and types of anions or cations are varied (6xLipid gymnastics: evidence of complete acyl chain reversal in oxidized phospholipids from molecular simulations. Khandelia, H. and Mouritsen, O.G. Biophys. J. 2009; 96: 2734–2743Abstract | Full Text | Full Text PDF | PubMed | Scopus (61)See all References6).The work of Knecht and Klasczyk (1xSpecific binding of chloride ions to lipid vesicles and implications at molecular scale. Knecht, V. and Klasczyk, B. Biophys. J. 2013; 104: 818–824Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References1), combining experimental work on electrophoresis and calorimetry with all-atom molecular dynamics calculations, suggests that force fields and the sampling of lipid and ionic interactions are still not sufficiently evolved to fully predict and interpret experimental measurements. The long-time and large-scale averages determined by experiment are not fully predicted and rationalized by the computations. Furthermore, as the authors point out, the simulations are in disagreement with each other, depending on force field, boundary conditions, and simulation size and time. Teasing out whether the issues are only force-field dependent, or also a function of sampling or system size, is a difficult issue that needs to be resolved.Further creating intrigue for our evolving understanding of the effects of charge on membrane function is the experimental findings that the size of the anion/cation matters. For example, the Hofmeister series has been found to be important as an organizing principle for the effects of anions in membrane behavior (7xEffects of monovalent anions of the Hofmeister series on DPPC lipid bilayers. Part I. Swelling and in-plane equations of state. Aroti, A., Leontidis, E...., and Zemb, T. Biophys. J. 2007; 93: 1580–1590Abstract | Full Text | Full Text PDF | PubMed | Scopus (56)See all References, 8xInfluence of anions and cations on the dipole potential of phosphatidylcholine vesicles: a basis for the Hofmeister effect. Clarke, R.J. and Lupfert, C. Biophys. J. 1999; 76: 2614–2624Abstract | Full Text | Full Text PDF | PubMedSee all References, 9xUnderstanding the Hofmeister effect in interactions between chaotropic anions and lipid bilayers: molecular dynamics simulations. Sachs, J.N. and Woolf, T.B. J. Am. Chem. Soc. 2003; 125: 8742–8743Crossref | PubMed | Scopus (92)See all References). If the electrostatic function alone were the dominant factor, then the type of anion would be much less important. So, finding out how and why size does play a role in the membrane behavior remains an intriguing area for research. This may also tie back into a need for improved force fields that can better predict the experimental results. It will be important to understand if the Hofmeister effects are related to binding, kinetics, or nonspecific screening effects.Gaining a molecular understanding of ion effects, even in water, has proven to be challenging from a molecular viewpoint (10xIon solvation thermodynamics from simulation with a polarizable force field. Grossfield, A., Ren, P.Y., and Ponder, J.W. J. Am. Chem. Soc. 2003; 125: 15671–15682Crossref | PubMed | Scopus (288)See all References10). Efforts to improve the current generation of force fields, supporting induced polarization, are encouraging and suggest that convergence in simulation results may be improved by the next generation of force fields (11xAbsolute hydration free energy scale for alkali and halide ions established from simulations with a polarizable force field. Lamoureux, G. and Roux, B. J. Phys. Chem. B. 2006; 110: 3308–3322Crossref | PubMed | Scopus (198)See all References11). These have yet to be fully applied to membrane simulations and the comparison of the results to earlier simulations and experiments will be interesting.For membrane protein function and the cellular biophysics of networks, these types of salt effects may also drive meaningful change. For example, if ion channels and ionic transport mechanisms are both directly and indirectly impacted by the distribution and types of charge, then this may play a role in their interconnections through the bilayer and may, in turn, change their functional specificity and kinetics. This implies that the behavior of membrane proteins and their associated connections to peptides, other proteins, and the bilayer environment, may be tied together with the seemingly straightforward, but still difficult, problem of how salt distributes in a membrane system.Thus, the article by Knecht and Klasczyk should be read as part of a continuing dialog on the interpretation of thermodynamic measurements at the molecular level and the role of simple systems in providing insights into the larger issues of cellular biophysics, membrane transport, and ionic interactions. It may presage the development of new insights into the physical properties of bilayers that update a view of dielectric screening based on boundary layers and nonspecific interactions with proteins and membrane headgroups.

The epidermal growth factor receptor (EGFR) is a major target for cancer drug development. Multiple x-ray structures provide some indications of the conformational changes that occur within the extracellular domain (ECD) upon activation by a growth factor ligand. These x-ray structures suggest a change at both the monomer level and at the dimer level. Yet the molecular details of how the conformations change with activation are not fully understood. We present calculations for the monomer of the transitions from a the tethered to an extended form of human EGFR, human ErbB2 and Drosophila EGFR. Our DIMS calculations for the dimer explore the recent asymmetric Drosophila dimer structure and create transitions from the symmetric to the asymmetric forms for the human EGFR and the Drosophila EGFR. The computations provide estimates of candidate reaction paths between the conformations as well as suggesting the effects of ligand binding and the transitions from the extracellular domains to the kinase activation.

All atom molecular dynamics is appealing due to the detailed insights towards protein function. But, all-atom molecular dynamics is limited due to the small amount of time spent in the transitions between stable states. Dynamic importance sampling (DIMS) enhances this sampling problem by biasing, with correction, to encourage sampling away from the stable states and to thus improve estimates of barrier crossing times. Nevertheless, DIMS has limitations based on the choice of order parameters that define intermediate states. To improve the representation, in DIMS, of order parameters, we have performed simulations of NtrC and have analyzed the transition populations. Our analysis, using techniques of nonlinear time series analysis and nonlinear dimensionality reduction, has enabled us to determine a new numerical method for finding reduced parameter descriptions of transitions in complex biomolecular systems. The results should enable an iterative approach to finding the best order parameter and at the same time improve convergence for estimates of reaction pathways, kinetics and relative free energy differences.

The sarcoplasmic reticulum (SR) ATPase (SERCA) actively transports calcium ions across the membrane. A number of sequential steps are involved in the catalytic cycle, starting with the binding of two Ca2+ ions to the ground state (E2) to form a phosphorylated intermediate (ADP.E1P.2Ca2+). ADP dissociation is followed by the isomerization of E1P.2Ca2+ to E2P.2Ca2+ and dissociation of Ca2+, finally hydrolytic cleavage of Pi from E2P. Twenty five mutations have been identified on the Actuator (A), Phosphorylation (P) and Nucleotide binding (N) domains that have significant impact on the structure and function of Ca-ATPase (Toyoshima et al, Biochemistry (2005), 44, 8090-8100). While a lot has been studied about the relative positions of domains and the structural changes involved in the catalytic cycle, the actual kinetics and conformational transitions are yet to be explored. The main focus of this research is to study the impact of these mutations on the kinetics of reactions involving conformational changes in the catalytic cycle of SERCA. Since this required generating many sets of transitions between intermediate states, we have implemented the coarse-grained protein and lipid model (Marrink et al, JCTC(2007) 4(5), 819-834) in CHARMM. Coarse-grained models have been used to address the problem of time scales inaccessible to the all atom approach. We use Dynamic Importance Sampling (DIMS) to generate transitions between the intermediate states. Transitions are generated between each mutated open and closed conformational state in both coarse-grained and all atom model in CHARMM. A comparison of both sheds light on the kinetics and the nature of transitions involving structural changes during the opening and closing of the pump.

Time-dependent red shift (TDRS) in the emission of Trp after impulse excitation reports the dynamics of solvent and protein relaxation. TDRS of the single Trp in the B1 domain of streptococcal protein G (GB1) has been measured experimentally [Toptygin, Gronenborn and Brand, J. Phys. Chem. B 2006, 110, 26292]. The present work compares the results of Charmm MD simulations with the experimental data. The protein in a box of explicit TIP3P water was equilibrated with the ground-state charges on Trp atoms. At t=0 the charges on Trp atoms were changed to those characteristic of the excited state 1La. TDRS was calculated from a series of 2000ps excited-state MD trajectories using first-order QM perturbation theory. TDRS from a single trajectory is overwhelmed by random noise. Averaging over 100 trajectories produced a result qualitatively consistent with the experimental data. The amplitudes of the calculated and measured TDRS during the time interval from 200ps to 2000ps are in good agreement. However, at t - 2 of random fluctuations in the ground-excited state energy gap ΔE and the relation hν(t)-hν(infinity)=C(t)/(kBT) [Nilsson and Halle, PNAS 2005, 102, 13867]. This produced a TDRS curve containing less high-frequency noise, but more low-frequency noise as compared to the result of averaging over 100 short nonequilibrium trajectories. We thank Patrick R. Callis for accurate excited-state Trp atom charges. Supported by NSF grants MCB-0416965, MCB-0719248.

The light-driven proton pump bacteriorhodopsin (bR) is a transmembrane protein that uses large conformational changes for proton transfer from the cytoplasmic to the extracellular regions. Crystal structures, due to their solvent conditions, do not resolve the effect of lipid molecules on these protein conformational changes. To begin to understand the molecular details behind such large conformational changes, we simulated two conformations of wild-type bacteriorhodopsin, one of the dark-adapted state and the second of an intermediate (M(O)) state, each within an explicit dimyristoyl-phosphatidylcholine (DMPC) lipid bilayer. The simulations included all-hydrogen and all-atom representations of protein, lipid, and water and were performed for 20 ns. We investigate the equilibrium properties and the dynamic motions of the two conformations in the lipid setting. We note that the conformational state of the M(O) intermediate bR remains markedly different from the dark-adapted bR state in that the M(O) intermediate shows rearrangement of the cytoplasmic portions of helices C, F, and G, and nearby loops. This difference in the states remained throughout the simulations, and the results are stable on the molecular dynamics timescale and provide an illustration of the changes in both lipid and water that help to stabilize a particular state. Our analysis focuses on how the environment adjusts to these two states and on how the dynamics of the helices, loops, and water molecules can be related to the pump mechanism of bacteriorhodopsin. For example, water generally behaves in the same manner on the extracellular sides of both simulations but is decreased in the cytoplasmic region of the M(O) intermediate. We suspect that the different water behavior is closely related to the fluctuations of microcavities volume in the protein interior, which is strongly coupled to the collective motion of the protein. Our simulation result suggests that experimental observation can be useful to verify a decreased number of waters in the cytoplasmic regions of the late-intermediate stages by measuring the rate of water exchange with the interior of the protein.

Intrinsically disordered proteins are an important class of proteins with unique functions and properties. Here, we have applied a support vector machine (SVM) trained on naturally occurring disordered and ordered proteins to examine the contribution of various parameters (vectors) to recognizing proteins that contain disordered regions. We find that a SVM that incorporates only amino acid composition has a recognition accuracy of 87 ± 2%. This result suggests that composition alone is sufficient to accurately recognize disorder. Interestingly, SVMs using reduced sets of amino acids based on chemical similarity preserve high recognition accuracy. A set as small as four retains an accuracy of 84 ± 2%; this suggests that general physicochemical properties rather than specific amino acids are important factors contributing to protein disorder.

Two concepts are presented for accurate nonequilibrium work free-energy measurements, realized both in molecular simulation and experiment. First, the need for an intermediate important to both the reference and the target systems (overlap) is indicated. Second, the use of a soft path from each end point to the intermediate (funnel) is demonstrated. Schemes implementing these concepts dramatically improve efficiency and accuracy of free energy calculations, as shown by calculation of the free energy of ion charging in water, and the free energy change in mutation of an adenosine molecule.

Recent X-ray structures of voltage gated potassium channels provide an exciting opportunity to connect molecular structures with measured biological function. Two of the most important connections for these channels are: first, to the molecular basis behind selectivity and the associated free energy profile underlying ionic current flow and, second, to a true molecular understanding of the large-scale conformational transitions that underlie voltage dependent gating. But, existing computational tools need to be further developed to reach these goals. In this contribution to the symposia on sampling methods we outline our dynamic importance sampling method for sampling large-scale conformational transitions as well as our studies with non-equilibrium work events and equilibrium overlap sampling (OS) methods for sampling events related to the calculation of relative free energies.

We present coarse-grained simulations of fusion between two liposomes from which a detailed picture of lipid movements emerges. In these simulations the bilayers dilate at the contact edge, and the resulting increase in the area per lipid produces a tilting of the individual molecules as predicted. Fusion is initiated when some of these tilted lipids splay their aliphatic tails, such that the molecules are shared between the opposing leaflets. Multiple splayed lipids subsequently associate with their aliphatic tails in contact, which produces a new hydrophobic core. As the tails extend into a more parallel conformation the two outer leaflets become contiguous to produce a hemifused structure. The results have interesting implications for biological membrane fusion and suggest new possibilities for designing molecules that control fusion.

Recent work from the Bos group has shown that the exchange protein activated by cAMP (EPAC), binds a single cAMP to allosterically regulate an ionic lock inducing a large conformational change and opening a binding site for the GTPases Rap1 or Rap2. Their x-ray structures of EPAC consist of two large regions with five domains. An ionic lock is defined by R886 and D883 of CDC25-HD and E332, D307 and Q303 of the CNB. When this lock is released the hinge region swings closer to the CNB domain creating a 45-Angstrom shift in the structural region. Mutations in this ionic lock (R886A) in EPAC produce a reduction in GEF activity, rather than an increase. This unexpected result is at odds with a simple picture of a direct tradeoff between cAMP binding energy and ionic lock release. DIMS trajectories of the conformational transition allow us to compute the energetic role of the mutation in entropy-enthalpy compensation and elucidate the allosteric communication pathways that are modulated by the mutation. Reference trajectories of the wild-type and the mutation (R886A) in the inactive and active states give us additional insights into the nature of the changes.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The regulation of copper levels is central to physiology. Mutations in the ATP7B copper transporter are known to lead to Wilson's disease in humans. How these mutations lead to the disease is not fully characterized at a molecular level. An excellent model system for exploring the changes in structure and dynamics for Wilson disease mutations for the ATP binding domain is provided by CopB from A. fulgidus. This domain has high sequence similarity with the P-, N-domains and hinge regions of ATP7B. Mutations to each region have previously been characterized by in vitro experimental measurements such as ATPase assays and intrinsic tryptophan fluorescence. In this presentation we highlight a net ∼5 μs of implicit and explicit solvent simulations conducted on the National supercomputer resource XSEDE (Stampede, Kraken, Keeneland and Lonestar) of the CopB wild-type and 13 Wilsons disease mutations found across each of these three regions. Solvent accessible surface area measurements, H-bonds analysis and schlitter entropy calculations showed that the mutations induced changes in the dynamics of the Closed conformations of CopB with respect to the Open structure revealing conformational transitions at different rates about the hinge region. While the mutations in the P and N-domains caused mild to moderate deviations with respect to the WT, the mutations in the Hinge region caused significant deviations. The results shed new light on how the disease mutations impact on conformational change, on ATP-binding, and on phosphorylation within these domains.

Fatty acid binding proteins (FABPs) can discriminate between saturated and unsaturated fatty acids via molecular mechanisms that are not understood. Molecular dynamics computer calculations are used to suggest the relationship between tertiary structure and binding specificity. Three separate 1-ns simulations, with explicit solvent, are presented: 1) oleic acid (C18:1 cis) bound to adipocyte FABP, 2) oleic acid bound to human muscle FABP, and 3) elaidic acid (C18:1 trans) bound to human muscle FABP. The average structural, dynamic, and energetic properties of the trajectory were analyzed, as were the motional correlations. The molecular dynamics trajectories reveal intriguing differences among all three systems. For example, the two proteins have different strengths of interaction energy with the ligand and different motional coupling, as seen with covariance analysis. This suggests distinctive molecular behavior of monounsaturated fatty acids in the two similar proteins. An importance scale, based on motional correlation and interaction energy between protein and ligand, is proposed, to help identify amino acids involved with the discrimination of ligand saturation state or geometric isomerization.

Despite significant advances in purification and in high-throughput experimental design, the assignment of ligands to orphaned odorant receptors is difficult. We describe the development of a three stage computational database that is coupled with experimental measurements to aid the de-orphanization process. We posit that apo receptor conformations can be found that rationalize binding and non-binding ligands first measured in experiment. The challenge is to find the best receptor models in this large set of candidates. Our approach is to filter the search space throughout each of our stages. The initial stage of database control begins with a set of tertiary models for the receptor made with Rosetta and Modeller. All-atom molecular dynamics is performed for the apo receptor in an explicit bilayer for each model. Using OpenEye's Fred and UCSF's Dock6, we determine the subset of sampled conformations that maximally rationalizes the current experiments. In a second stage, using the filtered set of reduced conformations we perform re-scoring with Poisson Boltzmann and re-evaluation relative to experiment. Our third stage uses Amber and Charmm potentials to further evaluate the quality of the reduced subset to fit against experiment. Lastly, new ligand computations with this reduced set of candidate receptor models are used to computationally screen for new ligands to be tested by experiment. A loop between experiment and computation is thus created, with any results feeding back into the database and multiple cycles used. We will emphasize results achieved on olfr1393 and olfr1392.

The microscopic details of lipid-protein interactions are examined using molecular dynamics simulations of the gramicidin A channel embedded in a fully hydrated dimyristoyl phosphatidylcholine (DMPC) bilayer. A novel construction protocol was used to assemble the initial configurations of the membrane protein complex for the simulations. Three hundred systems were constructed with different initial lipid placement and conformations. Seven systems were simulated with molecular dynamics. One system was simulated for a total of 600 psec, four were simulated for 300 psec, and two for 100 psec. Analysis of the resulting trajectories shows that the bulk solvent-membrane interface region is much broader than traditionally pictured in simplified continuum theories: its width is almost 15 angstroms. In addition, lipid-protein interactions are far more varied, both structurally and energetically, than is usually assumed: the total interaction energy between the gramicidin A and the individual lipids varies from 0 to -50 kcal/mol. The deuterium quadrupolar splittings of the lipid acyl chains calculated from the trajectories are in good agreement with experimental data. The lipid chains in direct contact with the GA are ordered but the effect is not uniform due to the irregular surface of the protein. Energy decompositions shows that the most energetically favorable interactions between lipid and protein involve nearly equal contributions from van der Waals and electrostatic interactions. The tryptophans, located near the bulk-membrane interface, appear to be particularly important in mediating both hydrogen bonding interactions with the lipid glycerol backbone and water and also in forming favorable van der Waals contacts with the hydrocarbon chains. In contrast, the interactions of the leucine residues with the lipids, also located near the interface, are dominated by van der Waals interactions with the hydrocarbon lipid chains.

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.

Dendritic spines are generally believed to play a role in modulating synaptically induced electrical events. In addition, they may also confine second messengers and thus topologically limit the distance over which second messenger cascades may be functionally significant. In order to address this possibility, computer simulations of transient second messenger concentration changes were performed. The results show the importance of spine morphology and binding and extrusion mechanisms in controlling second messenger transients. In the presence of intrinsic cytoplasmic binding sites and kinetic rates similar to that expected for calcium, second messengers were confined to the spine head. In the absence of binding/extrusion mechanisms, the size and time course of the input transient to the spine head influenced the second messenger transients that might be seen at the base of the spine neck and in other spines. Large and/or sustained increases in second messenger concentration in the spine head were communicated to the spine base and to other spine heads. The results emphasize the importance of a knowledge of breakdown pathways, concentrations and kinetics of binding sites, and extrusion mechanisms for understanding the dynamics of local chemical changes for dendritic spine function. © 1994 Wiley-Liss, Inc.

Major conformational changes are involved in the multi-step catalytic cycle of the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) pump. The movement and rotation of the Actuator (A) domain is crucial for ion translocation. The A-domain is connected to the trans-membrane helices through linker regions. Mutational studies on the A-M3 linker region show that varying the length of this region causes significant changes in the rate of the conformational transitions (JBC (2009),284,12258-12265 ). The focus of this research is to study the impact of these mutations on the structural changes during the transition from E1 to E2, the behavior of the A-M3 linker region, and the overall rate of the conformational transitions. In order to achieve faster computation of transitions, we implemented the MARTINI coarse-grained protein and lipid model in CHARMM. We used Dynamic Importance Sampling (DIMS) to compute transitions from the E1 to the E2 state for both directions of each mutant in both coarse-grained and all atom models. Analysis of the transitions across mutants shows that the angle formed by A-P-N domains changes by up to 20 degrees with an increasing number of inserts into the A-M3 linker region. Estimates of barrier crossing time from the simulation and experimental values are highly correlated (R2=0.934). Quasiharmonic analysis on the domains, linkers and transmembrane helices show entropic changes between the mutants and compensation effects. Interaction energies of the same regions indicate entropic-enthalpic compensation. Further investigation of the end state simulations shows changes in the number of high density water sites around the A-M3 linker region across the mutants. The varying degree of the change in volume due to water sites across all the mutants indicates a ripple effect where the local entropy-enthaply changes translate to global entropy-enthalpy changes.

The Time-Dependent Spectral Shifts (TDSS) in the fluorescence of solvatochromic dyes in polar solvents report solvent relaxation dynamics, which in water occurs on the femtosecond timescale. The TDSS in the emission of tryptophan and other solvatochomic fluorophores in proteins span a range of timescales from femtoseconds to nanoseconds. MD simulations of the GB1 protein in TIP3P water made it possible to separate five relaxation modes and to explain their physical origins. Two of these relaxation modes also contribute to the TDSS of dyes in polar solvents.The ultrafast relaxation mode (τ∼35fs) is due in part to the librational relaxation of water molecules and in part to the small adjustments in the local protein structure. This mode is responsible for about half of the total TDSS amplitude.Two collective rotational relaxation modes of water molecules are known. The longitudinal (τL∼550fs) mode contributes to the TDSS of both dyes in water and tryptophan residues in proteins. The transverse (τD∼8.3ps) relaxation cannot contribute to the TDSS of dyes in water, but it contributes to the TDSS in proteins having internal water channels or pockets. This can be used to study internal water. In MD simulations using TIP3P water both τL and τD are ∼30% shorter than the experimental values.A small (

Voltage-dependent potassium channels conduct potassium ions across the cell membrane by cycling between open and closed states. The transition between these well-defined states is driven by changes in the voltage drop across the bilayer. A growing dataset of K+ channel structures provides an opportunity to understand the molecular details of voltage-dependent gating. Determining the molecular details of gating is challenging because the x-ray structures are of either an open or closed state and of different sequence types. Thus, to gain an understanding of the molecular details of gating we are using dynamic importance sampling (DIMS) to create simulations of the transitions. Our starting point comes from the creation of a set of homology models for the full range of K+ channel sequences and structures that have been determined to date. Thus, we present simulations to suggest the transitions between open and closed states of four different sequences of K+ channels. The results suggest experimental alterations that influence gating and help to rationalize existing results. Furthermore the set of transitions suggests common modes to gating used in all K-channels.

We present all-atom molecular dynamics simulations of biologically realistic transmembrane potential gradients across a DMPC bilayer. These simulations are the first to model this gradient in all-atom detail, with the field generated solely by explicit ion dynamics. Unlike traditional bilayer simulations that have one bilayer per unit cell, we simulate a 170 mV potential gradient by using a unit cell consisting of three salt-water baths separated by two bilayers, with full three-dimensional periodicity. The study shows that current computational resources are powerful enough to generate a truly electrified interface, as we show the predicted effect of the field on the overall charge distribution. Additionally, starting from Poisson's equation, we show a new derivation of the double integral equation for calculating the potential profile in systems with this type of periodicity.

The position and extent of movement of a charged peptide within a membrane bilayer provides much controversy. In our study, we have examined the nature of the highly charged helix-turn-helix motif (S3b and S4) to address how a highly charged peptide is stabilized within a bilayer in the presence of various transmembrane electrical potentials. Our double-bilayer simulation results show how the variation of the salt concentrations between the inner and outer bath establishes a transmembrane potential. Our results also show that important features of the peptide affected by changes in electrical potential are the center of mass depth, the swivel/kink degrees of conformation, and the hydrogen-bonding patterns. As the voltage gradient across the bilayer increased, the center of mass of the peptide shifted in a direction toward the outer bath. The peptide also has a higher percent helical content and the swivel/kink conformation is more rigid for nonpolarized systems where no voltage drop occurred between salt baths. Our results also provide some suggestions for how this domain may be affected by environmental changes as part of the voltage sensor in a K-channel.

It is commonly believed that binding affinity can be estimated by consideration of local changes of ligand and protein. This paper discusses a set of molecular dynamics simulations of intestinal fatty acid binding protein addressing the protein's response to presence or absence of different ligands. A 5-ns simulation was performed of the protein without a ligand, and three simulations (one 5-ns and two 2-ns) were performed with different fatty acids bound. The results indicate that, although the basic protein structure is unchanged by the presence of the ligand, other properties are significantly affected by ligand binding. For example, zero-time covariance patterns between protein, bound waters, and ligand vary between the different simulations. Moreover, the interaction energies between ligand and specific residues indicate that different ligands are stabilized in different ways. In sum, the results suggest that binding thermodynamics within this system will need to be calculated not from a subset of nearby protein:ligand interactions, but will depend on a knowledge of the motions coupling together water, protein, and ligand.

Many proteins function as dynamic molecular machines that cycle between well-defined states. A mechanistic and atomic-scale understanding starts with crystal, NMR or electron microscopy structures in these states. Typically, none or only very limited structural information is available for the intermediates along the transition. Computational methods can simulate transitions between states but due to the absence of intermediate structures it is hard to verify that the simulated transition path is correct. One exception is the enzyme adenylate kinase. It is well studied and a large number of crystal structures are available. Vonrhein et al [1] suggested early on that some of these structures would be transition intermediates due to stabilization by crystal contacts and created a `movie' from nine structures. We took this idea one step further and compare 45 experimental structures to hundreds of transitions of E. coli AdK simulated with the dynamic importance sampling method (DIMS). We find that DIMS trajectories, which only require a crystal structure for the starting and the end point of the transitions, contain all intermediate crystal structures (RMSD for matches

Understanding and predicting conformational change in macromolecules is central to linking structure and function. Performing straight-forward all-atom molecular dynamics would, in principle, enable sampling of conformational changes. However, the time-scale for functionally important transitions, exceeds the usual molecular dynamics timescales by several orders of magnitude. For example, with large amounts of computer time all these transitions could be observed with good statistics and the results collected simply by waiting long enough. Thus to sample on longer time-scales requires the development of biased molecular dynamics methods, where the bias can be applied and corrected for at the end. In our approach, called ‘Dynamic Importance Sampling’ we generate a series of independent trajectories that are conditioned on starting and ending in defined conformations. Trajectories are generated using two different algorithms: one uses a soft-racheting scheme based on stochastic trajectories and the other uses information from the set of normal modes. The algorithms, which require no initial pathway, are capable of rapidly determining multiple pathways between known states. The associated probablity scores, determined by correcting for the bias, allows us to rank order the most likely pathways. We will present examples from three-helix bundles and other systems for both analysis and possible experimental work.

Sampling till completion is a new framework for conformational search that uses a coupled feedback between running computational dynamics and a large database. By using this feedback we can get improved convergence as well as more efficient sampling. We have applied this concept to sampling of peptide degrees of freedom and the first part of our presentation will highlight the challenges in making this idea work by addressing both hardware and software issues. This includes our efforts to create rapid query evaluations and our use of coupled CPU and GPU systems. The second part of our presentation will focus on the nature of the efficiency gains and how the concept may be helpful for sampling within the Onsager-Machlup framework for understanding conformational transitions. This enables us to view certain degrees of freedom as more important for the transitions than others and ties into dynamic importance sampling and effective transfer entropy approaches.

Improvements in sampling on the events leading to transitions can provide significant insights into what drives biomolecular change. We present additions to our Molecular Marshal (M2) software framework for the adaptive sampling of biomolecular transitions. As an example, we work with the transitions seen in BPTI from DEShaw Research (Science, 2010) using their long-running 1 ms trajectory as the basis for states and transitions analyzed from the Anton production trajectory. Our algorithm works by resampling snapshot conformations known to be right before transition events, creating an ensemble set of transitions preconditioned on sampling in the space near to the transitions. This enables us to explore the reduced degrees of freedom that drive the transitions and to examine the statistical foundations of the description for state transitions in BPTI. To achieve these goals we use an adaptive framework for resampling built around a parallel relational database system and with scripts controlling molecular dynamics codes running on XSEDE sponsored national supercomputers. In addition to BPTI, we will show results that start from other trajectories defined from peptides and from other long-running protein simulations. Our scripts for the initial analysis and the resampling thus readily generalize to both long and short trajectory runs and can be used to increase sampling on a broad range of transition events.

Proteins exist in aqueous solution. Hydration can be viewed as a description of how the protein disturbs the structure and dynamics of water. Water molecules in the vicinity of proteins are generally seen as either external or internal water molecules. Internal water molecules occupy cavities, exchange on a time-scale of 0.1--10 microseconds with bulk water, are almost as conserved as amino acids, and are therefore likely to be important for function. External water molecules tend to be found in protein crevices and are typically not conserved, even between crystal structures of the same protein. We introduce a method to analyze the behavior of water molecules in molecular dynamics (MD) simulations in terms of graphs. The graph encodes a simple hopping model: Nodes in the graph correspond to hydration sites, typically defined from the density in computer simulations or observed water sites in crystal structures. Directed edges correspond to transitions (“hops”) between sites, with transition rates computed from MD simulations. We apply this analysis to the water-filled cavity of intestinal fatty acid binding protein (I-FABP) in its apo and holo (palmitate-bound) state. This demonstrates how ligand binding influences the well-defined set of hydration sites in and around the protein's cavity. The ligand displaces a number of hydration sites but does not affect others close by. The parameters extracted from the network model allow us to model the movement of water molecules with a Markov Chain Monte Carlo model. The graphical construct reproduces the average site occupancy found in the MD simulations and the fluctuations of the occupancy.This approach suggests new types of sampling and analysis that can be applied to extend the range of molecular dynamics models and the role of water in ligand binding.

Improvements in sampling on the events leading to transitions can provide significant insights into what drives biomolecular change. We present additions to our Molecular Dynamics Database (MDDB). We start with the transitions seen in BPTI from DEShaw Research (Science, 2010) using their long-running 1 ms trajectory as the basis for a Markov chain with states and transitions analyzed from the Anton production trajectory. Our algorithm works by resampling snapshot conformations known to be right before transition events, creating an ensemble set of transitions preconditioned on sampling in the space near to the transitions. This enables us to explore the reduced degrees of freedom that drive the transitions and to examine the statistical foundations of the Markov chain description for state transitions in BPTI. To achieve these goals we use an adaptive framework for resampling built around a parallel relational database system and with scripts controlling molecular dynamics codes running on XSEDE sponsored national supercomputers. In addition to BPTI, we will show results that start from other trajectories defined from peptides and from other long-running protein simulations. Our scripts for the initial analysis and the resampling thus readily generalize to both long and short trajectory runs and can be used to increase sampling on a broad range of transition events.