Alan Grossfield | University of Rochester (original) (raw)
Papers by Alan Grossfield
Journal of the American Chemical Society, 2005
Biophysical Journal, 2014
Biophysical Journal, 2014
Biophysical Journal, 2014
Biophysical Journal, 2015
Biophysical Journal, 2014
Biophysical Journal, 2014
Journal of computational chemistry, Jan 15, 2014
LOOS (Lightweight Object Oriented Structure-analysis) is a C++ library designed to facilitate mak... more LOOS (Lightweight Object Oriented Structure-analysis) is a C++ library designed to facilitate making novel tools for analyzing molecular dynamics simulations by abstracting out the repetitive tasks, allowing developers to focus on the scientifically relevant part of the problem. LOOS supports input using the native file formats of most common biomolecular simulation packages, including CHARMM, NAMD, Amber, Tinker, and Gromacs. A dynamic atom selection language based on the C expression syntax is included and is easily accessible to the tool-writer. In addition, LOOS is bundled with over 140 prebuilt tools, including suites of tools for analyzing simulation convergence, three-dimensional histograms, and elastic network models. Through modern C++ design, LOOS is both simple to develop with (requiring knowledge of only four core classes and a few utility functions) and is easily extensible. A python interface to the core classes is also provided, further facilitating tool development.
a matter of debate if the translocation route taken by colicin N is through the OmpF internal por... more a matter of debate if the translocation route taken by colicin N is through the OmpF internal pore or via the external protein-lipid interface. Recent electron microscopy data from our laboratory suggests the latter route for translocation [2]. In order to re-address this question we undertook small-angle neutron scattering experiments. By using a combination of deuterated OmpF and hydrogenated colicin N we were able to define the three dimensional structure of the colicin N-OmpF complex. This revealed that colicin N inserts into clefts on the outside of the OmpF trimer, supporting the case for translocation via the proteinlipid interface. 1. el Kouhen, R., et al., Eur J Biochem, 1993. 214(3) 2. Baboolal, T. G., et al., Structure, 2008. 16(3) Fig1. The ab initio SANS model of OmpF (light grey) and colicin N (dark grey) complex.
The universal response to stress is mediated by the expression of heat shock proteins (hsp), whic... more The universal response to stress is mediated by the expression of heat shock proteins (hsp), which are involved in repair and recovery from the insult. In addition, their presence provides protection from subsequent insults, which has been coined stress tolerance. The cytoprotective role of hsp has been associated with their chaperone function within the cytosol. However, hsp were recently found outside cells where they acted as signaling molecules directed at activating the immune system to avoid the propagation of the insult. Hsp70, the major inducible form of the hsp family, does not contain any consensus secretory signal that predicts its secretion via the ER-Golgi pathway. Therefore, it is likely that the export of Hsp70 to the extracellular environment is mediated by the propose that Hsp70 is exported by a novel mechanism initiated by the translocation of the protein into the plasma membrane and is released associated with vesicles called export or extracellular vesicles (ECV). To test this hypothesis, we developed a liposome insertion assay using pure recombinant Hsp70. We found that Hsp70 insertion into lipid membranes was spontaneous and specific for negatively charged lipids, such as phosphatidylserine and phosphatidylglycerol. In contrast, positive or neutral lipids did not support membrane insertion. We also found that less fluid lipid environments highly favored membrane insertion, which resembles in vivo observations indicating the presence of Hsp70 in lipid rafts of cells. In summary, our observations support the hypothesis that membrane insertion is the first stage of secretion of Hsp70 into the extracellular environment in the form of vesicles. We speculate that hsp70-ECV may result in a robust activation of the immune system that is part of the systemic response to stress. Synaptotagmin (Syt) proteins serve as Ca 2þ sensitive triggers in many exocytotic pathways, with the seventeen different human isoforms active in various cell types. Syt proteins contain two C2 domains, C2A and C2B, which bind membranes in response to Ca 2þ to drive vesicle fusion. Much is known about the biophysical mechanism of function for Syt1, which triggers fusion for rapid neurotransmitter secretion, but less mechanistic information is available for other isoforms. Syt7 typically operates in slower pathways requiring smaller peak Ca 2þ concentrations, and its C2 domains are known to bind membranes with a much higher Ca 2þ sensitivity compared to Syt1. using kinetic and equilibrium fluorescence measurements of C2A domain docking to synthetic liposomes approximating the lipid composition of physiological membranes, we report that the differences between the two isoforms include kinetic and solute effects consistent with much greater hydrophobic membrane contact for Syt7 C2A. A strong hydrophobic contribution to the membrane docking mechanism of Syt7 C2A stands in contrast to the known electrostatic membrane interaction of Syt1 C2A, and is somewhat surprising given the 90% conserved amino acid polarity between the two domains. In order to test our proposed hydrophobic docking model for Syt7 C2A and probe its structural origins, we use a combination of site-directed mutagenesis, equilibrium and kinetic protein-membrane docking assays, and electron paramagnetic resonance-based depth measurements. In addition, single-molecule measurement of protein lateral diffusion on supported lipid bilayers is used to report on contributions of intra-and intermolecular protein-protein contact to the membrane-docked states of individual C2 domains and C2AB tandems. The results are interpreted to provide information on the structural origins of differences in function between these two isoforms. The GPCRs b2 Adrenergic Receptors and Histamine Receptors have been shown to have extracellular enhancement of their receptor activity by ascorbate. Ascorbate binds on the 2 nd extracellular loop and shifts the agonist dose-response curve approximately 0.5 log units to the left. When present with ascorbate, receptor-membrane preparations significantly reduced the rate of ascorbate oxidation. As the molar concentration of ascorbate greatly exceeded that of the receptors, one-to-one binding is not possible, and the presences of receptor reductase activity was suspected. In the studies below, ascorbate but not its oxidation product DHA is shown to absorb at 265 nm using both UV spectroscopy (UVS) and capillary electrophoresis (CE). Ascorbate oxidation is shown by the disappearance of ascorbate absorbance. DHA reduction is shown by the appearance of ascorbate. The equilibrium constant for DHA/Asc was found to be 49: 10 mM DHA produced 20 mM Asc at equilibrium. using UVS (AR) and CE (HR), both b2AR and HR increase the DHA reduction rate by 40-50% at 20 mM phosphate. The rate of DHA reduction was found to be dependent on both the phosphate concentration and specifically on the presence of HPO 4 2-. The net reduction of DHA is DHA þ 2 HPO 4 2-4 Asc þ 2 PO 4 3with the reaction enzymatically enhanced by b2AR and HR. At physiological phosphate concentrations of 1 mM, the fractional increase in DHA reduction due to GPCRs may be even greater than that measured at 20 mM phosphate, as the reduction rate for 1 mM phosphate alone is very low. These observations may be an important addition to GPCR function and to evolutionary theories of reduction in the pre-biotic world. Enthalpic and entropic changes during GPCR activation are poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several 'local activating switches' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain composed of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the b2 adrenergic receptor from molecular dynamics simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm in terms of receptor entropic maps provides a novel way to design functionally biased mutants and may also assist in the identification of allosteric sites in GPCRs.
Rhodopsin is currently one of two proteins of the G protein-coupled receptor (GPCR) family for wh... more Rhodopsin is currently one of two proteins of the G protein-coupled receptor (GPCR) family for which an atomic-resolution structure is available and therefore it serves as a prototypical GPCR. Over 50% of recently launched drugs are targeted to GPCRs so that these proteins are of outstanding pharmaceutical interest. Yet many aspects of the structure-function relations of rhodopsin are only poorly understood. Especially interesting is the important role played by highly unsaturated lipids in achieving full functionality. In this study we investigated lipid-protein interactions of rhodopsin through a series of molecular dynamics (MD) simulations. Rhodopsin was inserted into a membrane consisting of a 2:2:1 mixture of 18:0,22:6n3PE, 18:0,22:6n3PC, and cholesterol. MD simulations with a total trajectory of~4 microseconds were conducted on the IBM Blue Gene/L supercomputer. Additional simulations were carried out for rhodopsin in membranes consisting of 18:0,18:1PC and 14:0,14:1PC. This allowed direct comparison of the lipid-protein interactions of rhodopsin in monounsaturated and polyunsaturated environments as well as bilayers of varying thickness. Our analysis focuses on the palmitoylations of two adjacent cysteins in helix 8 of rhodopsin which are a common structural motif in many proteins related to signal transduction. As seen in our earlier studies on lipid modifications of smaller proteins, these are generally highly flexible and best characterized by their dynamical structure. We calculated order parameters and correlation functions from the simulation trajectories and compared them to experimentally obtained results on lipid modifications of other proteins. In general we find the that the palmitoyl modifications of rhodopsin are highly flexible which can affect the binding free energy of the polypeptide to the membrane surface. Entropic contributions due to the configurational disorder of the posttranslational modifications reduce the binding energy so that doubly palmitoylated modifications are needed. The dynamics of biological membranes is present on many time-and length scales, from the pico-second vibrations of single lipid tail groups to micro-second collective undulations of hundreds of lipid molecules. These motions can be probed as local density fluctuations, and characterized and measured in different ways. Inelastic scattering experiments as well as computer simulations of biological membranes are usually theoretically interpreted within the framework of generalized hydrodynamics, where the dynamic structure factor, S(k,w), is the quantity of interest. S(k,w) is the space and time Fourier transform of the density-density correlation function and contains all the relevant information about the dynamics of a liquid system. We have performed largescale molecular dynamics simulations from which we have calculated the dynamic structure factors for a lipid bilayer in the high temperature phase, enabling a thorough test of theoretical predictions, especially in the hydrodynamic limit of low wave vectors. The frequency and wave vector resolutions are considerably improved compared to traditional experiments, which make it possible to directly resolve the lines of the power spectrum. Membrane material constants have been determined and compared in fair agreement to experimental data. In addition, from the power spectrum we can distinguish two dispersive contributions to the elastic scattering. These correspond to two exponential relaxation processes on separate time scales. For low wave vectors, this analysis was impossible due to insufficient frequency resolution. To be able to fully decouple and analyze the nature of these dispersive modes from simulations, longer trajectories are required.
several new algorithms and implementation techniques that enable the detection of significant str... more several new algorithms and implementation techniques that enable the detection of significant structure-changing events in a molecular dynamics trajectory. These algorithms include a coarse graining of side chain contacts, a contact metric based on higher-order generalizations of the Delaunay tetrahedralization, and median filters for detecting significant shifts in the ensemble mean of the resulting time series. We have also developed numerical techniques for suppressing trivial re-crossing events and a new kernel-based estimator of the contact alteration activity. These methods will be disseminated in a newly developed package, ''TimeScapes,'' which is compatible with molecular dynamics trajectories generated from any of a variety of popular simulation programs. Tests on microsecond time scale simulations suggest that the implementation is efficient and requires very little parameterization. The analysis provides a detailed listing of broken and formed contacts, and reliably detects allosteric and folding transitions, as well as stable intermediates, in the protein dynamics.
Rhodopsin is currently the only available atomic-resolution template for understanding biological... more Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the β-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the β-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The β-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state 2 H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.
Biophysical Journal, 2014
The development of novel antibiotic drugs is one of the most pressing biomedical problems due to ... more The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK's binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK's selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.
determined that prestin diffuses in and hops between confinement zones on the order of 1 micron i... more determined that prestin diffuses in and hops between confinement zones on the order of 1 micron in average size. We observe that depletion of membrane cholesterol increases average confinement size and decreases confinement strength. Statistical analysis of squared displacements reveals that depletion of cholesterol removes domains of intermediate size between 142 and 500 nm. From measurements of the initial increase of the mean squared deviation with time, the microscopic diffusion constant was determined to be 0.05 X 10 À12 m 2 /s, and was unchanged by cholesterol depletion. Our results suggest that membrane cholesterol affects prestin function by changing prestin crowding in confinement zones, consistent with the hypothesis that the microscale organization of prestin in the membrane influences prestin function.
Journal of the American Chemical Society, 2005
Biophysical Journal, 2014
Biophysical Journal, 2014
Biophysical Journal, 2014
Biophysical Journal, 2015
Biophysical Journal, 2014
Biophysical Journal, 2014
Journal of computational chemistry, Jan 15, 2014
LOOS (Lightweight Object Oriented Structure-analysis) is a C++ library designed to facilitate mak... more LOOS (Lightweight Object Oriented Structure-analysis) is a C++ library designed to facilitate making novel tools for analyzing molecular dynamics simulations by abstracting out the repetitive tasks, allowing developers to focus on the scientifically relevant part of the problem. LOOS supports input using the native file formats of most common biomolecular simulation packages, including CHARMM, NAMD, Amber, Tinker, and Gromacs. A dynamic atom selection language based on the C expression syntax is included and is easily accessible to the tool-writer. In addition, LOOS is bundled with over 140 prebuilt tools, including suites of tools for analyzing simulation convergence, three-dimensional histograms, and elastic network models. Through modern C++ design, LOOS is both simple to develop with (requiring knowledge of only four core classes and a few utility functions) and is easily extensible. A python interface to the core classes is also provided, further facilitating tool development.
a matter of debate if the translocation route taken by colicin N is through the OmpF internal por... more a matter of debate if the translocation route taken by colicin N is through the OmpF internal pore or via the external protein-lipid interface. Recent electron microscopy data from our laboratory suggests the latter route for translocation [2]. In order to re-address this question we undertook small-angle neutron scattering experiments. By using a combination of deuterated OmpF and hydrogenated colicin N we were able to define the three dimensional structure of the colicin N-OmpF complex. This revealed that colicin N inserts into clefts on the outside of the OmpF trimer, supporting the case for translocation via the proteinlipid interface. 1. el Kouhen, R., et al., Eur J Biochem, 1993. 214(3) 2. Baboolal, T. G., et al., Structure, 2008. 16(3) Fig1. The ab initio SANS model of OmpF (light grey) and colicin N (dark grey) complex.
The universal response to stress is mediated by the expression of heat shock proteins (hsp), whic... more The universal response to stress is mediated by the expression of heat shock proteins (hsp), which are involved in repair and recovery from the insult. In addition, their presence provides protection from subsequent insults, which has been coined stress tolerance. The cytoprotective role of hsp has been associated with their chaperone function within the cytosol. However, hsp were recently found outside cells where they acted as signaling molecules directed at activating the immune system to avoid the propagation of the insult. Hsp70, the major inducible form of the hsp family, does not contain any consensus secretory signal that predicts its secretion via the ER-Golgi pathway. Therefore, it is likely that the export of Hsp70 to the extracellular environment is mediated by the propose that Hsp70 is exported by a novel mechanism initiated by the translocation of the protein into the plasma membrane and is released associated with vesicles called export or extracellular vesicles (ECV). To test this hypothesis, we developed a liposome insertion assay using pure recombinant Hsp70. We found that Hsp70 insertion into lipid membranes was spontaneous and specific for negatively charged lipids, such as phosphatidylserine and phosphatidylglycerol. In contrast, positive or neutral lipids did not support membrane insertion. We also found that less fluid lipid environments highly favored membrane insertion, which resembles in vivo observations indicating the presence of Hsp70 in lipid rafts of cells. In summary, our observations support the hypothesis that membrane insertion is the first stage of secretion of Hsp70 into the extracellular environment in the form of vesicles. We speculate that hsp70-ECV may result in a robust activation of the immune system that is part of the systemic response to stress. Synaptotagmin (Syt) proteins serve as Ca 2þ sensitive triggers in many exocytotic pathways, with the seventeen different human isoforms active in various cell types. Syt proteins contain two C2 domains, C2A and C2B, which bind membranes in response to Ca 2þ to drive vesicle fusion. Much is known about the biophysical mechanism of function for Syt1, which triggers fusion for rapid neurotransmitter secretion, but less mechanistic information is available for other isoforms. Syt7 typically operates in slower pathways requiring smaller peak Ca 2þ concentrations, and its C2 domains are known to bind membranes with a much higher Ca 2þ sensitivity compared to Syt1. using kinetic and equilibrium fluorescence measurements of C2A domain docking to synthetic liposomes approximating the lipid composition of physiological membranes, we report that the differences between the two isoforms include kinetic and solute effects consistent with much greater hydrophobic membrane contact for Syt7 C2A. A strong hydrophobic contribution to the membrane docking mechanism of Syt7 C2A stands in contrast to the known electrostatic membrane interaction of Syt1 C2A, and is somewhat surprising given the 90% conserved amino acid polarity between the two domains. In order to test our proposed hydrophobic docking model for Syt7 C2A and probe its structural origins, we use a combination of site-directed mutagenesis, equilibrium and kinetic protein-membrane docking assays, and electron paramagnetic resonance-based depth measurements. In addition, single-molecule measurement of protein lateral diffusion on supported lipid bilayers is used to report on contributions of intra-and intermolecular protein-protein contact to the membrane-docked states of individual C2 domains and C2AB tandems. The results are interpreted to provide information on the structural origins of differences in function between these two isoforms. The GPCRs b2 Adrenergic Receptors and Histamine Receptors have been shown to have extracellular enhancement of their receptor activity by ascorbate. Ascorbate binds on the 2 nd extracellular loop and shifts the agonist dose-response curve approximately 0.5 log units to the left. When present with ascorbate, receptor-membrane preparations significantly reduced the rate of ascorbate oxidation. As the molar concentration of ascorbate greatly exceeded that of the receptors, one-to-one binding is not possible, and the presences of receptor reductase activity was suspected. In the studies below, ascorbate but not its oxidation product DHA is shown to absorb at 265 nm using both UV spectroscopy (UVS) and capillary electrophoresis (CE). Ascorbate oxidation is shown by the disappearance of ascorbate absorbance. DHA reduction is shown by the appearance of ascorbate. The equilibrium constant for DHA/Asc was found to be 49: 10 mM DHA produced 20 mM Asc at equilibrium. using UVS (AR) and CE (HR), both b2AR and HR increase the DHA reduction rate by 40-50% at 20 mM phosphate. The rate of DHA reduction was found to be dependent on both the phosphate concentration and specifically on the presence of HPO 4 2-. The net reduction of DHA is DHA þ 2 HPO 4 2-4 Asc þ 2 PO 4 3with the reaction enzymatically enhanced by b2AR and HR. At physiological phosphate concentrations of 1 mM, the fractional increase in DHA reduction due to GPCRs may be even greater than that measured at 20 mM phosphate, as the reduction rate for 1 mM phosphate alone is very low. These observations may be an important addition to GPCR function and to evolutionary theories of reduction in the pre-biotic world. Enthalpic and entropic changes during GPCR activation are poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several 'local activating switches' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain composed of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the b2 adrenergic receptor from molecular dynamics simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm in terms of receptor entropic maps provides a novel way to design functionally biased mutants and may also assist in the identification of allosteric sites in GPCRs.
Rhodopsin is currently one of two proteins of the G protein-coupled receptor (GPCR) family for wh... more Rhodopsin is currently one of two proteins of the G protein-coupled receptor (GPCR) family for which an atomic-resolution structure is available and therefore it serves as a prototypical GPCR. Over 50% of recently launched drugs are targeted to GPCRs so that these proteins are of outstanding pharmaceutical interest. Yet many aspects of the structure-function relations of rhodopsin are only poorly understood. Especially interesting is the important role played by highly unsaturated lipids in achieving full functionality. In this study we investigated lipid-protein interactions of rhodopsin through a series of molecular dynamics (MD) simulations. Rhodopsin was inserted into a membrane consisting of a 2:2:1 mixture of 18:0,22:6n3PE, 18:0,22:6n3PC, and cholesterol. MD simulations with a total trajectory of~4 microseconds were conducted on the IBM Blue Gene/L supercomputer. Additional simulations were carried out for rhodopsin in membranes consisting of 18:0,18:1PC and 14:0,14:1PC. This allowed direct comparison of the lipid-protein interactions of rhodopsin in monounsaturated and polyunsaturated environments as well as bilayers of varying thickness. Our analysis focuses on the palmitoylations of two adjacent cysteins in helix 8 of rhodopsin which are a common structural motif in many proteins related to signal transduction. As seen in our earlier studies on lipid modifications of smaller proteins, these are generally highly flexible and best characterized by their dynamical structure. We calculated order parameters and correlation functions from the simulation trajectories and compared them to experimentally obtained results on lipid modifications of other proteins. In general we find the that the palmitoyl modifications of rhodopsin are highly flexible which can affect the binding free energy of the polypeptide to the membrane surface. Entropic contributions due to the configurational disorder of the posttranslational modifications reduce the binding energy so that doubly palmitoylated modifications are needed. The dynamics of biological membranes is present on many time-and length scales, from the pico-second vibrations of single lipid tail groups to micro-second collective undulations of hundreds of lipid molecules. These motions can be probed as local density fluctuations, and characterized and measured in different ways. Inelastic scattering experiments as well as computer simulations of biological membranes are usually theoretically interpreted within the framework of generalized hydrodynamics, where the dynamic structure factor, S(k,w), is the quantity of interest. S(k,w) is the space and time Fourier transform of the density-density correlation function and contains all the relevant information about the dynamics of a liquid system. We have performed largescale molecular dynamics simulations from which we have calculated the dynamic structure factors for a lipid bilayer in the high temperature phase, enabling a thorough test of theoretical predictions, especially in the hydrodynamic limit of low wave vectors. The frequency and wave vector resolutions are considerably improved compared to traditional experiments, which make it possible to directly resolve the lines of the power spectrum. Membrane material constants have been determined and compared in fair agreement to experimental data. In addition, from the power spectrum we can distinguish two dispersive contributions to the elastic scattering. These correspond to two exponential relaxation processes on separate time scales. For low wave vectors, this analysis was impossible due to insufficient frequency resolution. To be able to fully decouple and analyze the nature of these dispersive modes from simulations, longer trajectories are required.
several new algorithms and implementation techniques that enable the detection of significant str... more several new algorithms and implementation techniques that enable the detection of significant structure-changing events in a molecular dynamics trajectory. These algorithms include a coarse graining of side chain contacts, a contact metric based on higher-order generalizations of the Delaunay tetrahedralization, and median filters for detecting significant shifts in the ensemble mean of the resulting time series. We have also developed numerical techniques for suppressing trivial re-crossing events and a new kernel-based estimator of the contact alteration activity. These methods will be disseminated in a newly developed package, ''TimeScapes,'' which is compatible with molecular dynamics trajectories generated from any of a variety of popular simulation programs. Tests on microsecond time scale simulations suggest that the implementation is efficient and requires very little parameterization. The analysis provides a detailed listing of broken and formed contacts, and reliably detects allosteric and folding transitions, as well as stable intermediates, in the protein dynamics.
Rhodopsin is currently the only available atomic-resolution template for understanding biological... more Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the β-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the β-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The β-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state 2 H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.
Biophysical Journal, 2014
The development of novel antibiotic drugs is one of the most pressing biomedical problems due to ... more The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK's binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK's selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.
determined that prestin diffuses in and hops between confinement zones on the order of 1 micron i... more determined that prestin diffuses in and hops between confinement zones on the order of 1 micron in average size. We observe that depletion of membrane cholesterol increases average confinement size and decreases confinement strength. Statistical analysis of squared displacements reveals that depletion of cholesterol removes domains of intermediate size between 142 and 500 nm. From measurements of the initial increase of the mean squared deviation with time, the microscopic diffusion constant was determined to be 0.05 X 10 À12 m 2 /s, and was unchanged by cholesterol depletion. Our results suggest that membrane cholesterol affects prestin function by changing prestin crowding in confinement zones, consistent with the hypothesis that the microscale organization of prestin in the membrane influences prestin function.