Use of fluorescent probes in the study of phospholipid--sterol bilayers.
Ontology highlight
ABSTRACT:
SUBMITTER: Wharton SA
PROVIDER: S-EPMC1162278 | biostudies-other | 1980 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Nearest-neighbor recognition experiments have been carried out in fluid liposomal membranes made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol using exchangeable dimers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and cholesterol. In cholesterol-rich bilayers, the association between these two exchangeable lipids was reduced as the curvature of the membrane increases; that is, when the diameter of the liposomes was below ca. 200 nm. In sharp contrast, the mixing of these exchangeable lipids was close to random in the cholesterol-poor membranes, regardless of their curvature. The biological implications of these findings are briefly discussed.
| S-EPMC2674620 | biostudies-literature
Project description:Exploring the detailed structural features of synthetic molecules in the membrane phase requires sensitive probes of conformation. Here we describe the design, synthesis and characterization of bis(pyrene) probes that report conformational changes in membrane-active dynamic foldamers. The probes were designed to distinguish between left-handed (M) and right-handed (P) screw-sense conformers of 310-helical α-aminoisobutyric acid (Aib) peptide foldamers, both in solution and in bilayer membranes. Several different bis(pyrene) probes were synthesized and ligated to the C-terminus of Aib tetramers that had different chiral residues at the N-terminus, residues that favored either an M or a P screw-sense in the 310-helix. The readily synthesized and conveniently incorporated N-acetyl-1,2-bis(pyren-1'-yl)ethylenediamine probe proved to have the best properties. In solution, changes in foldamer screw-sense induced substantial changes in the ratio of excimer/monomer fluorescence emission (E/M) for this reporter of conformation, with X-ray crystallography revealing that opposite screw-senses produce very different interpyrene distances in the reporter. In bilayers, this convenient and sensitive fluorescent reporter allowed, for the first time, an investigation of how the chirality of natural phospholipids affects foldamer conformation.
| S-EPMC6114994 | biostudies-literature
Project description:Lipid bilayers provide the structural framework for cellular membranes, and their character as two-dimensional fluids enables the mobility of membrane macromolecules. Though the existence of membrane fluidity is well established, the nature of this fluidity remains poorly characterized. Three-dimensional fluids as diverse as chocolates and cytoskeletal networks show a rich variety of Newtonian and non-Newtonian dynamics that have been illuminated by contemporary rheological techniques. Applying particle-tracking microrheology to freestanding phospholipid bilayers, we find that the membranes are not simply viscous but rather exhibit viscoelasticity, with an elastic modulus that dominates the response above a characteristic frequency that diverges at the fluid-gel (L(α) - L(β)) phase-transition temperature. These findings fundamentally alter our picture of the nature of lipid bilayers and the mechanics of membrane environments.
| S-EPMC2984223 | biostudies-literature
Project description:Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.
| S-EPMC7745015 | biostudies-literature
Project description:The spontaneous self-assembly of ?-synuclein (?-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the ?-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that ?-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of ?-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of ?-syn. Interaction of ?-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of ?-syn with membranes plays a critical role initiating the formation of ?-syn aggregates and the overall aggregation process.
| S-EPMC6661114 | biostudies-literature
Project description:Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.
| S-EPMC2836762 | biostudies-literature
Project description:The emerging crisis of antibiotic resistance requires a multi-pronged approach in order to avert the onset of a post-antibiotic age. Studies of antibiotic uptake and localisation in live cells may inform the design of improved drugs and help develop a better understanding of bacterial resistance and persistence. To facilitate this research, we have synthesised fluorescent derivatives of the macrolide antibiotic erythromycin. These analogues exhibit a similar spectrum of antibiotic activity to the parent drug and are capable of labelling both Gram-positive and -negative bacteria for microscopy. The probes localise intracellularly, with uptake in Gram-negative bacteria dependent on the level of efflux pump activity. A plate-based assay established to quantify bacterial labelling and localisation demonstrated that the probes were taken up by both susceptible and resistant bacteria. Significant intra-strain and -species differences were observed in these preliminary studies. In order to examine uptake in real-time, the probe was used in single-cell microfluidic microscopy, revealing previously unseen heterogeneity of uptake in populations of susceptible bacteria. These studies illustrate the potential of fluorescent macrolide probes to characterise and explore drug uptake and efflux in bacteria.
| S-EPMC8341779 | biostudies-literature
Project description:A growing body of literature suggests that fluorocarbons can direct self-assembly within hydrocarbon environments. We report here the fabrication and characterization of supported lipid bilayers (SLBs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a synthetic, fluorocarbon-functionalized analogue, 1. AFM investigation of these model membranes reveals an intricate, composition-dependent domain structure consisting of approximately 50 nm stripes interspersed between approximately 1 microm sized domains. Although DSC of 1 showed a phase transition near room temperature, DSC of DPPC:1 mixtures exhibited complex phase behavior suggesting domain segregation. Finally, temperature-dependent AFM of DPPC:1 bilayers shows that, while the stripe structures can be melted above the Tm of 1, the stripes and domains result from immiscibility of the hydrocarbon and fluorocarbon lipid gel phases. Fluorination appears to be a promising strategy for chemical self-assembly in two dimensions. In particular, because no modification is made to the lipid headgroups, it may be useful for nanopatterning biologically relevant ligands on bilayers in vitro or in living cells.
| S-EPMC2507729 | biostudies-literature
Project description:A nearest-neighbor recognition analysis has been performed in cholesterol-rich and cholesterol-poor liposomes derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of varying concentrations of chloroform. This analysis has yielded a fundamentally new, molecular-level view of the interaction of general anesthetics with lipid bilayers, which may be relevant to their biological action; that is, DPPC forms 1:1 complexes with CHCl(3) in both membranes in the fluid bilayer state.
| S-EPMC2856843 | biostudies-literature
Project description:Despite significant efforts and promising progress, the understanding of membrane protein folding lags behind that of soluble proteins. Insights into the energetics of membrane protein folding have been gained from biophysical studies in membrane-mimicking environments (primarily detergent micelles). However, the development of techniques for studying the thermodynamics of folding in phospholipid bilayers remains a considerable challenge. We had previously used thiol-disulfide exchange to study the thermodynamics of association of transmembrane alpha-helices in detergent micelles; here, we extend this methodology to phospholipid bilayers. The system for this study is the homotetrameric M2 proton channel protein from the influenza A virus. Transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. Thiol-disulfide exchange under equilibrium conditions was used to quantitatively measure the thermodynamics of this folding interaction in phospholipid bilayers. The effects of phospholipid acyl chain length and cholesterol on the peptide association were investigated. The association of the helices strongly depends on the thickness of the bilayer and cholesterol levels present in the phospholipid bilayer. The most favorable folding occurred when there was a good match between the width of the apolar region of the bilayer and the hydrophobic length of the transmembrane helix. Physiologically relevant variations in the cholesterol level are sufficient to strongly influence the association. Evaluation of the energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclusion of cholesterol in the lipid bilayers.
| S-EPMC299801 | biostudies-literature