1. Membrane Partitioning/Membrane Binding Binding to Proteins: Specific well defined binding sites Number of binding sites per molecule Strength of the interaction cooperativity Adsorption 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 attraction Different Models of Binding: Partition Equilibrium Langmuir Adsorption Isotherms Complexation to N Lipids

2. What do we mean by BINDS?

3. Partition Equilibrium P + L  PL P = small molecule (protein etc) L = lipid, lipid is considered a separate phase Partitioning Coefficient: Kp = Cb/Cf Where 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 constant

4. Langmuir Adsoprtion Isotherm: membrane is treated as a lattice of potential binding sites – often not relevant to membrane binding phenomenon Complexation to n-lipids L + nP LPn L = ligand, small molecule P = phospholipid Ka = [LPn] / ([L][P] n ) --- resulting equations are referred to as Scatchard Plots Specific Ligand Interactions, Electrostatics, Dehydration all play a role in binding

5. Classes 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: Ions Class I: Benzene, hydrocarbons, perfloroalkanes PFOB Nonpolar molecules

7. Antimicrobial Peptides Antibiotics/Antifungals Many 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 permeabilization Mode of interaction is based upon specific properties of the peptide and target membrane BBA 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 peptides Lytic peptides: eukaryotic, prokaryotic, both

8. Peptide-Membrane Interactions: membrane-peptide mediated : can change sterochemistry and still have lytic effects, in fact, gramicidin is composed of altering L and D amino acids Most anit bacterial peptides contain high charge and amphipathic nature Hemolytic peptides : low net positive or negative charge. Bee Venom : Mellitin Frog toxin: Meganin Antifungals: Nystatin and Amphotericin B (polyene macrolide) Fungal and mammallian cells – recognize sterols, more strongly ergosterol than cholesterol (basis for fungal selectivity)

9. 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 monomers

11. Detergent interactions with Bilayers: last week lecture and presentation. Small Probe Molecule: EPR and Fluorescent Molecules Membrane “Fluidity” Measured by TEMPO partitioning f = fraction in the bilayer Hyperfine splitting is sensitive to polarity H is “bilayer” P is aqueous buffer Shows Gel to liquid transition

12. Bilayer Permeability: (a) enter membrane, (b) diffuse across (c)exit membrane P = KpDm/d Kp = Cm/Caq