Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions (original) (raw)
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Biophysical Journal, 2010
Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids-an insight with significant implications for theoretical description of bilayer lipid dynamics.
Single-Molecule Fluorescence Imaging of Peptide Binding to Supported Lipid Bilayers
Analytical Chemistry, 2009
Single-molecule fluorescence imaging techniques have been adapted to the quantitative characterization of peptide-binding to lipid bilayers. Peptide-membrane interactions are important in therapeutics, diagnostics, and membrane permeation and for understanding of the structure and function of membrane-bound proteins. Total-internal reflection fluorescence (TIRF) imaging is capable of determining membrane-binding equilibrium constants through the reliable counting of individual peptide molecules in order to report their surface density in the membrane. The residence times of the individual molecules in the membrane can also be determined and the rates of unbinding determined from a histogram of residence times. A combination of the unbinding kinetics and the equilibrium constant allows the binding rate of a peptide to the membrane also to be reported. We apply this method to characterize the lipid membrane affinity of glucagon-like peptide-1 (GLP-1), a 30-residue membrane-active peptide that is involved in glycemic control. Using single-molecule TIRF imaging, we have measured the affiliation of GLP-1 with a supported, phospholipid bilayer and determined its binding equilibrium constant. Two rates of dissociation were observed, suggesting strongly and weakly bound states of the peptide. The rate of membrane association was much slower than diffusioncontrolled, indicating a significant kinetic barrier to membrane binding. The data were interpreted using a heterogeneous, surface-reaction model analogous to electron-transfer kinetics at an electrode. To our knowledge, these results are the first example of using single-molecule counting to quantify peptide-lipid bilayer binding equilibria and kinetics.
Biophysical Journal, 2010
Direct visualization of the mechanism(s) by which peptides induce localized changes to the structure of membranes has high potential for enabling understanding of the structure-function relationship in antimicrobial and cell-penetrating peptides. We have applied a combined imaging strategy to track the interaction of a model antimicrobial peptide, PFWRIR-IRR-amide, with bacterial membrane-mimetic supported phospholipid bilayers comprised of POPE/TOCL. Our in situ studies revealed rapid reorganization of the POPE/TOCL membrane into localized TOCL-rich domains with a concomitant change in the organization of the membranes themselves, as reflected by changes in fluorescent-membrane-probe order parameter, upon introduction of the peptide.
Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers
Proceedings of the National Academy of Sciences, 2013
Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.
Single-molecule microscopy on model membranes reveals anomalous diffusion
Biophysical Journal, 1997
The lateral mobility of lipids in phospholipid membranes has attracted numerous experimental and theoretical studies, inspired by the model of Singer and Nicholson (1972. Science, 175:720-731) and the theoretical description by Saffman and Delbrück (1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113). Fluorescence recovery after photobleaching (FRAP) is used as the standard experimental technique for the study of lateral mobility, yielding an ensemble-averaged diffusion constant. Single-particle tracking (SPT) and the recently developed single-molecule imaging techniques now give access to data on individual displacements of molecules, which can be used for characterization of the mobility in a membrane. Here we present a new type of analysis for tracking data by making use of the probability distribution of square displacements. The potential of this new type of analysis is shown for single-molecule imaging, which was employed to follow the motion of individual fluorescence-labeled lipids in two systems: a fluid-supported phospholipid membrane and a solid polymerstabilized phospholipid monolayer. In the fluid membrane, a high-mobility component characterized by a diffusion constant of 4.4 microns2/s and a low-mobility component characterized by a diffusion constant of 0.07 micron2/s were identified. It is proposed that the latter characterizes the so-called immobile fraction often found in FRAP experiments. In the polymer-stabilized system, diffusion restricted to corrals of 140 nm was directly visualized. Both examples show the potentials of such detailed analysis in combination with single-molecule techniques: with minimal interference with the native structure, inhomogeneities of membrane mobility can be resolved with a spatial resolution of 100 nm, well below the diffraction limit.
J. Am. Chem. …, 2009
Helix-helix association within a membrane environment represents one of the fundamental processes in membrane protein folding. However, studying the kinetics of such processes has been difficult because most membrane proteins are insoluble in aqueous solution. Here we present a stopped-flow fluorescence study of the membrane interaction kinetics of a designed, water-soluble transmembrane (TM) peptide, anti-α IIb , which is known to dimerize in phospholipid bilayers. We show that by using two fluorescent amino acids, i.e., tryptophan and p-cyano-phenylalanine, we are able to kinetically dissect distinct phases in the peptide-membrane interaction, representing membrane binding, membrane insertion, and TM helix-helix association. Our results further show that the latter process occurs on a time scale of seconds, indicating that the association of two TM helices is an intrinsically slow event.
Membranes, 2015
Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * s-1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D sup or approximatively 1um2 * s-1 for FCS and D inf or approximatively 5 um2 s-1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusio...
Tracking individual membrane proteins and their biochemistry: The power of direct observation
Neuropharmacology, 2015
The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing t...
Quantitative visualization of passive transport across bilayer lipid membranes
Proceedings of the National Academy of Sciences, 2008
The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest tha...
Biophysical Journal, 2006
The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-b-maltoside or dodecyl-b-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20°C and at 4°C with three bacterial photosynthetic multisubunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of twodimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.