Abstract:Biological membranes are composed of lipid bilayers that are often asymmetric with regards to the lipid composition and/or aqueous solvent they separate. Studying lipid asymmetry both experimentally and computationally is challenging. Molecular dynamics simulations of lipid bilayers with asymmetry are difficult due to finite system sizes and time scales accessible to simulations. Due to the very slow flip-flop rate for phospholipids, one must first choose how many lipids are on each side of the bilayer, but th… Show more
“…The Coulomb interactions were calculated using the smooth particle–mesh Ewald method (Cerutti et al, 2009 ; Kratz et al, 2016 ; Boateng, 2020 ) with a Fourier grid spacing of 0.12 nm (Fischer et al, 2015 ). The simulation in the NpT ensemble was achieved by semi-isotropic coupling at 1 bar with coupling constants of 4 ps (Aoki and Yonezawa, 1992 ; Blumer et al, 2020 ) and temperature-coupling the simulation system using velocity Langevin dynamics with a coupling constant of 1 ps (Washio et al, 2018 ). The integration time step was 2 fs.…”
We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (CLas) proteins, LasP235 and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these CLas proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC–MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP235 and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP235 and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to CLas lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP235 and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP235and rescued the bactericidal activity of LTP thereby proving that LasP235, indeed, participates in HLB pathogenesis.
“…The Coulomb interactions were calculated using the smooth particle–mesh Ewald method (Cerutti et al, 2009 ; Kratz et al, 2016 ; Boateng, 2020 ) with a Fourier grid spacing of 0.12 nm (Fischer et al, 2015 ). The simulation in the NpT ensemble was achieved by semi-isotropic coupling at 1 bar with coupling constants of 4 ps (Aoki and Yonezawa, 1992 ; Blumer et al, 2020 ) and temperature-coupling the simulation system using velocity Langevin dynamics with a coupling constant of 1 ps (Washio et al, 2018 ). The integration time step was 2 fs.…”
We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (CLas) proteins, LasP235 and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these CLas proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC–MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP235 and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP235 and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to CLas lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP235 and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP235and rescued the bactericidal activity of LTP thereby proving that LasP235, indeed, participates in HLB pathogenesis.
“…The number of lipid molecules (128 total) was chosen based on recent reports. [65][66][67] Starting coordinates of the lipid bilayer, composed of 128 molecules of DMPC, were taken from the website of Biocomputing laboratory at the University of Calgary (http:// wcm.ucalgary.ca/tieleman/downloads) and solvated with 5227 water molecules in a rectangular box of 6.5 × 6.5 × 7.0 nm 3 . The mixed bilayers of DMPC and gemini components were obtained by a random selection of 52 molecules of DMPC replaced by the same number of gemini molecules.…”
Lipid stereochemistry was shown to drive cell internalization of liposomes through either clathrin or caveolae. Molecular dynamic simulations highlighted molecular interactions that affect physicochemical and biological features of liposomes.
“…Published results from MD simulations of asymmetric bilayers include investigations of the lateral organization of lipids within the two leaflets of asymmetric bilayers [79,80,84,85], the effects of asymmetry on dipole potential [5,21,86], cholesterol distribution [87,88], permeation [89], peptide-induced membrane deformation [39], and interleaflet coupling [79,[90][91][92][93], to name a few. The properties of any simulated bilayer are subject to the underlying set of force field parameters, which determine the forces exerted on the individual atoms and thus the dynamics of the system.…”
Section: Molecular Dynamics (Md) Simulations Used To Model Asymmetric Membranesmentioning
It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid–lipid and lipid–protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry.
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