Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Membrane Partitioning/Membrane BindingBinding to Proteins: •Specific well defined binding sites•Number of binding sites per molecule•Strength of the interaction•cooperativityAdsorption of Ions and Amphipathic molecules to bilayers is not always as straightforward – binding site may not be as well defined. There may be specific ligands, there may be hydrophobic partitioning, or electrostatic attractionDifferent Models of Binding:Partition EquilibriumLangmuir Adsorption IsothermsComplexation to N LipidsWhat do we mean by BINDS?Partition EquilibriumP + L PLP = small molecule (protein etc)L = lipid, lipid is considered a separate phasePartitioning Coefficient: Kp = Cb/CfWhere Cb is the concentration of bound molecule (PL), Cf is the concentration of free molecule in solution (P).Binding Isotherms are typically analyzed by measuring either the amount of the free ligand in solution or that bound to the bilayer and knowing the total concentration of lipid.An expression was given in Fridays paper presentation:Typically you derive an expression in terms of known total amounts, measure one parameter to determine a binding constantLangmuir Adsoprtion Isotherm: membrane is treated as a lattice of potential binding sites – often not relevant to membrane binding phenomenonComplexation to n-lipidsL + nP LPnL = ligand, small moleculeP = phospholipidKa = [LPn] / ([L][P]n) --- resulting equations are referred to as Scatchard PlotsSpecific Ligand Interactions, Electrostatics, Dehydration all play a role in bindingClasses of Ligands Which Interact with the Lipid Bilayer•Class I :Non-Polar Solutes•Class II: Amphipathic Molecules•Anesthetics - •Drugs – antipsychotics, antianxiety etc - chlorpromazine•Antibiotics•Detergents•Membrane Probes•Class III: Hydrophobic Ions•Class IV: IonsClass I: Benzene, hydrocarbons, perfloroalkanesPFOBNonpolar moleculesAntimicrobial Peptides Antibiotics/AntifungalsMany microbial antibiotics are peptides that form cationic amphipathic secondary structures that interact with negatively charged bacterial membranes via aid of electrostatic interactions. – form pores, leading to membrane permeabilizationMode of interaction is based upon specific properties of the peptide and target membraneBBA 1999 1462, issues 1-2•Amphipathic/hydrophobic -helices-magainin-sheet peptides and small proteins-defensins•Peptides with irregular AA composition-•Peptides with thio-ether rings-lantibiotics•Peptaibols –alamethicin Aib•Macrocyclic cysteine knot peptidesLytic peptides: eukaryotic, prokaryotic, bothPeptide-Membrane Interactions:membrane-peptide mediated : can change sterochemistry and still have lytic effects, in fact, gramicidin is composed of altering L and D amino acidsMost anit bacterial peptides contain high charge and amphipathic natureHemolytic peptides : low net positive or negative charge.Bee Venom : MellitinFrog toxin: MeganinAntifungals: Nystatin and Amphotericin B (polyene macrolide)Fungal and mammallian cells – recognize sterols, more strongly ergosterol than cholesterol (basis for fungal selectivity)•Only in contact with the head groups•Secondary structure unimportant•Not inserted into hydrophobic core•Monomers bind in -helical state•Monomers recognize and assemble•Helices insert into hydrophobic core•Additional recruitment of monomersDetergent interactions with Bilayers: last week lecture and presentation.Small Probe Molecule: EPR and Fluorescent MoleculesMembrane “Fluidity” Measured by TEMPO partitioningf = fraction in the bilayerHyperfine splitting is sensitive to polarityH is “bilayer”P is aqueous bufferShows Gel to liquid transitionBilayer Permeability: (a) enter membrane, (b) diffuse across (c)exit membraneP = KpDm/dKp =
View Full Document
Unlocking...