Lipids Membranes Lipids Classes

Lipids: Classes Biological molecules that are characterized by low solubility in water, that is, are relatively hydrophobic. Classes of Lipids They have a high hydrocarbon content 1. Fatty acids 2. Fats (triglycerides) 3. Waxes 4. Membrane Lipids 5. Isoprenes 2 & 3 are sometimes classified together as ”simple lipids” 1 Lipids: Fatty Acids (D9) (D9) (D9,12) (D9,12,15) (D6,9,12) (D5,8,11,14) (D15) Need to Know: Common names, structure, symbol (e.g., 18:3D9,12,15) Lipids: Membrane Lipids Classification of Membrane Lipids Two major categories based on the structure and function: 1. Lipids that contain phosphate 2. Lipids that do not contain phosphate – each can be further separated into: • Glycerol-based and sphingosine-based (animals) Cholesterol Sphingolipids Sphingophospholipids Sphingoglycolipids PS, PE, PC sphingomyelin 2 Lipids: Membranes Introduction The 4 S’s Size Solubility Shape Stability Models for Membrane structure Old Model Data Fluid Mosaic Model Testing the model The Red-Blood Cell Membrane Membrane Asymmetry Lipids transverse lateral Protein anchoring glycoproteins Membrane Fluidity Lipids: Membranes Models for Membrane Structure OLD MODEL (ca. 1940-1970) Sandwich model proposed by DanielliDavson. Based on the structures in the EM Bilayer Membrane proteins (mostly have b-structure) Scientifically, this is a good MODEL because it is clearly TESTABLE! This model makes several testable predictions: 1) Protein-lipid interactions should be mostly electrostatic; proteins should have lots of charged groups. 2) Should be able to “wash” nearly all membrane proteins off the membranes with high salt. 3) Isolated membrane proteins should show lots of b-structure 4) Importantly, NO PROTEINS ON THE INSIDE 3 Lipids: Membranes Models for Membrane Structure TESTING OLD MODEL: DATA 1) & 2) Wash isolated membranes with high-salt solutions or changes in pH. Ø Removes some but not all proteins Ø This leads to an operational definition of peripheral (those that wash off with 0.5 M salt), an integral (those that remain after washing) membrane proteins M 0.5 Cl Na det erg ent s Integral Peripheral d&c a, b, & e Amphitrophic and GPI-linked proteins Lipids: Membranes Models for Membrane Structure TESTING OLD MODEL: DATA 3) Isolated membrane proteins should show lots of b-structure. Ø Peripheral membrane proteins looked like cytosolic proteins Ø CD showed there was actually more ahelix than b-structure Ø Integral membrane proteins had patches of hydrophobic residues in their sequence 4 Lipids: Membranes Models for Membrane Structure TESTING OLD MODEL: DATA 4) Importantly, NO PROTEINS ON THE INSIDE. Ø So, lets look: performed Freeze-fracture EM on cell membranes Ø This immediately became an explanation for Integral membrane proteins. OMG!!! NOT smooth inside! Oops, maybe proteins DO span the membrane. (1000 Å) Lipids: Membranes Models for Membrane Structure NEW MODEL (1972) Fluid Mosaic Model proposed by SJ Singer & GL Nicholson • Lipids form a viscous, twodimensional solvent into which proteins are inserted and integrated more or less deeply. • Proteins can either be embedded in or associated with the membrane: SJ Singer (1924-2017) – Integral membrane proteins are firmly associated with the membrane, often spanning the bilayer. – Peripheral membrane proteins are weakly associated and can be removed easily. o Some are non-covalently attached. o Some are linked to membrane lipids (amphitrophic)(more later). This model was also testable! 5 Lipids: Membranes Testing Fluid Mosaic Model of Membrane Structure: Cellular Fusion Lipids: Membranes Testing Fluid Mosaic Model of Membrane Structure: FRAP • Fluorescence Recovery After Photobleaching (FRAP) allows us to monitor lateral diffusion by monitoring the rate of fluorescence return. • From the rate of return of fluorescently labeled lipids, the rate of diffusion of a lipid in the leaflet can be determined. Distance = √4Dt Set distance by radius of laser target area, measure t, calculate D D is rate of diffusion in m2/sec • Rates of lateral diffusion are high (up to 1 µm2/sec). – A lipid can circumnavigate an E.coli cell in one second. – A protein has a variable rate: 0.4 to 0.0001 µm2/sec. …..... but not so fast! 6 Lipids: Membranes Testing Fluid Mosaic Model of Membrane Structure: FRAP Lateral Movement is fast 2 A Single Lipid in 56 ms Transverse Movement is SLOW Spontaneous flips from one leaflet to another are rare because the charged head group must transverse the hydrophobic tail region of the membrane. Lipids: Membranes The Fluid Mosaic Model: Details 7 Lipids: Membranes Introduction The 4 S’s Size Solubility Shape Stability Models for Membrane structure Old Model Data Fluid Mosaic Model Testing the model The Red-Blood Cell Membrane Membrane Asymmetry Lipids transverse lateral Protein anchoring glycoproteins Membrane Fluidity Lipids: Membranes The Structure of the Red-blood Cell Membrane Lets look closer are one of these integral membrane proteins, glycophorin. 8 Lipids: Membranes The Structure of the Red-blood Cell Membrane Glycophorin Nonpolar Amino Acids of Integral Membrane Proteins are within the Membrane Lipids: Membranes Introduction The 4 S’s Size Solubility Shape Stability Models for Membrane structure Old Model Data Fluid Mosaic Model Testing the model The Red-Blood Cell Membrane Membrane Asymmetry Lipids transverse lateral Protein anchoring glycoproteins Membrane Fluidity 9 Lipids: Membranes Asymmetry • Membranes are very asymmetric. • All kinds of asymmetry: Components–lipids, proteins Types–transverse, lateral – Lipids (transverse): • Two leaflets have different lipid compositions. • The outer leaflet is often more positively charged. Net Charge: Rat liver plasma membrane 0 0 –1 0 • If Phosphatidylserine is found outside, it has a special meaning: – platelets: activates blood clotting – other cells: marks the cell for destruction How is this asymmetry maintained? Lipids: Membranes Asymmetry – Lipids (transverse): Flippases • Special enzymes catalyze transverse diffusion. – Though often referred to by category name “flippase,” there are unique unidirectional and bidirectional enzymes to catalyze lipid movement. • Some flippases use energy of ATP to move lipids against the concentration gradient. There exits lateral asymmetry; these enzymes determine and 10 Lipids: Membranes Asymmetry Lipids (lateral): 1) On the inner leaflet, can induce phosphoserine to coalesce with calcium. 2) On the outerleaflet, can induce “raft” formation; the coalescence of particular membrane lipids (cholesterol, sphingoglycolipids, sphingomyelin, etc.) Lipids: Membranes Asymmetry Lipids (lateral): Membrane Rafts Lipid distribution in a single leaflet is not random or uniform. Lipid rafts: • contain clusters of sphingoglycolipids with longer-than-usual tails and cholesterol • are more ordered (not as fluid) • contain specific doubly or triply acylated proteins • allow segregation of proteins in the membrane 11 Lipids: Membranes Asymmetry Lipids (lateral): Membrane Rafts Lipids: Membranes Asymmetry Proteins: 1) anchored 2) glycoproteins Ø Some membrane proteins are lipoproteins containing a covalently linked lipid molecule. Ø The lipid part inserts into the membrane. Ø The protein is then anchored to the membrane. Ø These are the aforementioned “Amphitropic” membrane proteins • This allows targeting of proteins to the membrane, either internally (FA or isoprenes), or externally (GPIlinked). • Some, such as GPI anchors are found only on the outer face of plasma membrane. • This is a reversible process. 12 Lipids: Membranes Asymmetry Proteins: 1) anchored 2) glycoproteins Ø Some membrane proteins are lipoproteins containing a covalently linked lipid molecule. Ø The lipid part inserts into the membrane. Ø The protein is then anchored to the membrane. Ø These are the aforementioned “Amphitropic” membrane proteins • This allows targeting of proteins to the membrane, either internally (FA or isoprenes), or externally (GPIlinked). • Some, such as GPI anchors are found only on the outer face of plasma membrane. • This is a reversible process. Lipids: Membranes Asymmetry Proteins: 1) anchored Found ONLY ever on the OUTSIDE of Cells 2) glycoproteins 13 Lipids: Membranes Introduction The 4 S’s Size Solubility Shape Stability Models for Membrane structure Old Model Data Fluid Mosaic Model Testing the model The Red-Blood Cell Membrane Membrane Asymmetry Lipids transverse lateral Protein anchoring glycoproteins Membrane Fluidity Lipids: Membranes Fluidity Membrane Phase Transition • Cells must maintain fluidity for membranes to function properly. • Depending on their composition and the temperature, the lipid bilayer must maintain a fluid phase; if cooled, it undergoes a phase transition and goes to a gel state. – liquid-ordered state (i.e., “gel phase”): individual molecules do not move around – NOT ACTIVE – liquid-disordered (or liquid crystal) state (i.e., “fluid phase”): individual molecules can move around – ACTIVE NOT ACTIVE Cooling ACTIVE NOT ACTIVE ACTIVE Semi-solid Fluid 14 Lipids: Membranes Fluidity Membrane Phase Transition Heating Conformations Conformations ACTIVE NOT ACTIVE Semi-solid Fluid Gel Liquid crystal Temperature Plot membrane thickness vs. temperature Lipids: Membranes Fluidity Membrane Phase Transition Normalized Membrane Thickness ACTIVE Fluid NOT ACTIVE Semi-solid Phase-transition point, or melting temperature Cooperativity is modified by cholesterol 15 Lipids: Membranes Fluidity Membrane Phase Transition Cholesterol Increases Membrane Rigidity and Permeability cholesterol HO • • Due to its small polar head, it lies deeper in the bilayer than the phospholipids Cell membranes of many eukaryotes contain sterols. – cholesterol in animals – phytosterols in plants – ergosterol in fungi • Cell membranes of aerobic prokaryotes contain hopanols. Lipids: Membranes Fluidity Organisms Can Adjust the Temperature of the Phase Transition by Changing the Membrane Composition • Membrane fluidity is determined mainly by the fatty acid composition and melting point. • The temperature of the phase transition (Tm): – Melting temperature higher with more saturated fatty acids. – Melting temperature higher with longer fatty acids. – Melting temperature lower with more unsaturated fatty acids. – Melting temperature lower with shorter fatty acids. • Therefore, at higher temperatures, cells need more long, saturated fatty acids. • And at lower temperatures, cells need more shorter, unsaturated fatty acids. 16 Lipids: Membranes Fluidity TABLE 11-2 Fatty Acid Composition of E. coli Cells Cultured at Different Temperatures Percentage of total fatty acidsa 10 ˚C 20 ˚C 30 ˚C 40 ˚C Myristic acid 4 4 4 8 Palmitic acid 18 25 29 48 Palmitoleic acid 26 24 23 9 Oleic acid 38 34 30 12 Hydroxymyristic acid 13 10 10 8 Ratio of unsaturated to saturatedb 2.9 2.0 1.6 0.38 SOURCE: Data from A. G. Marr and J. L. Ingraham, J. Bacteriol. 84:1260, 1962. a The exact fatty acid composition depends not only on growth temperature but on growth stage and growth medium composition. b Ratios calculated as the total percentage of 16:1 plus 18:1 divided by the total percentage of 14:0 plus 16:0. Hydroxymyristic acid was omitted from this calculation. 17