Membranes What are the purposes of membranes? Physical barriers/compartmentalization Gatekeepers—exclusion of toxic molecules Energy and signal transduction Aid in cell locomotion Cell-cell interactions

Lipid and Protein Concentration Vary in Different Types of Membranes Depending on the function of the membrane (structural vs. a place where reactions are catalyzed), the protein and lipid content changes Membranes that carry out enzyme reactions, signal transduction, transport, etc. are typically protein rich Lipid composition also varies

Lipids Order in Aqueous Solutions Amphipathic lipid form a variety of structures in water RAPIDLY. Micelles—lipid polar head faces outward, hydrophobic tails are inward. Typically made of a few hundred molecules. Critical Micelle Concentration: concentration of lipids needed to form micelles. Below CMC—individual lipids

Ordered Structures of Lipids - Bilayers form spontaneously over large areas

Bilayers minimize solvent exposure by wrapping around themselves—lamellar structures

Membrane Thickness and Shape Typical cell membranes are approximately 50Å thick (includes the lipid bilayer and membrane proteins) Thickness is defined by particular lipid/protein composition of the membrane – Some membrane proteins stick far out of the lipid bilayer and can change thickness by as much as 5Å! Though we draw lipids tails as being perpendicular to the membrane plane, they can actually tilt/bend (think about all those unsaturated fatty acids and their particular conformations!)

Fluid Mosaic Model Discovered by SJ Singer and GL Nicolson in 1972 Membranes are dynamic structures composed of proteins and phospholipids Phospholipid bilayer is a fluid matrix—2D solvent for proteins. 2 types of proteins in bilayer: – Peripheral/Extrinsic proteins • don’t penetrate bilayer • associated by non-covalent interactions to surface of bilayer or embedded proteins • dissociate by changing salt concentration/pH – Integral/Intrinsic proteins • have hydrophobic surfaces that penetrate bilayer and regions that interact with the aqueous environment

1 Cholesterol Impacts Membrane Fluidity—Prevents Extremes -OH group on cholesterol interacts with polar head groups, steroid/hydrocarbon chain buried in the lipid bilayer Decreases membrane fluidity, increases membrane packing—also prevents membrane crystallizaton Reduces the membrane’s permeability to neutral solutes, protons, and other ions—a good thing!

Membranes are More Mosaic than Fluid!

Peripheral MembraneProteins LOOSELY associated with membrane via 4 different types of interactions:

Integral Membrane Proteins: Single Transmembrane Segments Proteins anchored in membrane by a single hydrophobic segment—typically an α-helix. 10-30% of transmembrane proteins have a single helical transmembrane segment Typically function as cell surface receptors for extracellular signaling or immune recognition sites

Example: Glycophorin A Spans red blood cell membrane Glycoprotein—oligosaccharides dictate MN antigenic specificities

Integral Membrane Proteins: Multiple Transmembrane Segments Cross the lipid bilayer more than once Typically have 2-12 transmembrane segments Example: Bacteriorhodopsin • light driven proton transport for Halobacterium • Primary sequence: 7 segments about 20 nonpolar residues in length

Hydropathy Plots - Give us clues to determine whether or not a polypeptide sequence with a given hydrophobicity would likely be included in a membrane Beyond the α-helix: β-strands span the membrane too! β-barrels 2 – Maximize H-bonding, very stable – Interior can accommodate H2O molecules and peptide chains – Polar and non-polar residues alternate along the strands, polar face in (to interact w/ water), nonpolar face out and interact with lipids β-barrel Examples Porins: Maltoporin – transports maltose through E. coli outermembrane

Multiple Subunit β-barrel: α-hemolysin toxin (7-mer) – secreted as monomers by Staph. aureus – Channel allows uncontrolled permeation of water, ions, small molecules Membranes are Both Heterogeneous and Asymmetric Lateral heterogeneity—certain types of proteins and lipids cluster together in the membrane plane Transverse asymmetry—the inner and outer leaflet of a membrane may have different protein and lipid compositions Example: Typical animal cell—amine-containing phospholipids enriched in the cytoplasmic (inner) leaflet of the plasma membrane, and choline containing phospholipids and sphingolipids enriched in outer leaflet Imbalances in these levels and loss of transverse asymmetry can result in catastrophe—cell death signaling Moving Lipids Modulate Membrane Functions Lateral diffusion of lipids can occur quickly (could move in a linear direction several microns per sec.) Proteins can help move lipids from one side of the bilayer to the other – Flippases—ATP dependent, specific lipids – Floppases—ATP dependent, specific lipids – Scramblases—ATP independent, non-specific, requires Ca2+

Flippases, Floppases and Scramblases Help Maintain Transverse Asymmetry Membrane Ordering

Membrane Ordering Low Temps—gel phase/solid-ordered state, acyl chain perpendicular to membrane plane, chains packed, very little movement Higher Temps— liquid crystalline/liquid-disordered state, acyl chains in motion-rotation around C-C bonds and increased disorder

Moving between these two states is called a phase transition, and occurs at a transition temperature/melting temperature Sharpness of transition indicative of cooperative behavior—molecules in same vicinity acting in concert

3 Membrane Rafts The liquid ordered state—acyl chain ordering, but translational disorder Up to 50% of the plasma membrane consists of these rafts Small and transient—hard to measure! Hopping the Fence Kusumi et al. determined using single particle tracking of fluorescently labeled lipids that lipids tend to stay in compartments, but occasionally “hop over the fence” to new compartments.

Transport Across Membranes Passive Diffusion – Uncharged Molecule: Entropic process, movement across membrane happens until concentration of molecules the same on either side – Charged Molecule: Dependent on electrochemical potential

Transport Across Membranes FACILITATED DIFFUSION: Passive diffusion is too slow to sustain most biochemical processes

Facilitated diffusion occurs via proteins with net movement of solvent happening in a thermodynamically favored direction (dG < 0) These proteins typically have a high binding affinity for certain solutes

Membrane Channel Proteins Single Channel Pores: Made up of dimer, trimer, etc. multimeric protein subunits :

Multimeric subunit assemblies where each subunit has its own pore:

Facilitated Diffusion Membrane Channel Proteins Channels are often selective for a particular type of ion or molecule. – Usually have their pores lined with amino acids of the opposite charge of the ion they are transporting Some are gated—open/close upon a signal. – Voltage-gated respond w/ change in membrane potential. – Ligand gated open/close with binding of a specific ion, molecule, small protein Depending on channel size, ions can flow through in a hydrated or dehydrated state – Wide, non-selective – Narrow, charged amino acids lining, highly selective

Example: Potassium Channels Potassium transport is critical for maintaining cell volume, electrical impulse formation (neurons) K+ channels are highly selective for K+ over Na+, and conduct K+ ions at very fast rates—at the diffusion limits For the tetramer channel KcsA, four pentapeptides ThrValGlyTyrGly (backbone carbonyls and Thr oxygens) mediate selectivity of K+ vs Na+ – Selectivity based on atomic radius! Facilitated Transport: Potassium Channels High selectivity and quick transport—seems paradoxical! Repulsion from closely spaced K+ ions and conformational changes induced by binding keep things moving…. Channel Conformational Changes Leave the Channel Open or Closed pH induced helix bending and rearrangement

4 Active Transport Active Transport—transport of species from low to high concentrations—requires energy input – Energy sources come from ATP hydrolysis, light energy and energy stored in ion gradients. Monvalent cation transport coupled to ATP-hydrolysis – Na+,K+-ATPase—sodium pump

Na+,K+-ATPase Integral membrane protein with 3 subunits – alpha (120 kDa), beta (35 kDa), gamma (6.5 kDa) Actively pumps 3 Na+ out of cell and 2 K+ into cell for every one ATP hydrolyzed Na+,K+-ATPase Inhibitors

Cardiac glycosides/Cardiotonic steroids – Bind to extracellular side of Na+,K+-ATPase to form very stable complex

People with high blood pressure/hypertension have high levels of these inhibitors.

Accumulation of Na+ and Ca2+ narrows the vessels and creates hypertension

ABC Transporters ATP-Binding Cassette (ABC) transporters. Superfamily of transporters found across most ancient and modern cells. - Active transporters which use ATP to drive compound transport against gradient. - Transport ions, lipids, foreign compounds even nucleotides and membranes ABC transporters (over 1,100 different ABC transporters across all organisms / 50 in humans) – 2 transmembrane domains, form a pore; cytosolic nucleotide binding domains – Conserved core of 12 transmembrane helices - Mechanism of transport changes depending on specific transporter - 15 different known defects altering 14 of the 50 human transporters lead to known diseases - Multidrug resistance (MDR) efflux pumps (over expressed in some tumor cells) - export cellular waste molecules and toxins - can be a problem for certain therapeutic drugs - Other diseases associated with ABC transporters include: Cystic fibroses, age related macrodegeneration, and others 5