Dysfunction in Ganglioside Recycling Leads to a Variety of Medical Disorders

Nucleic Acids A. B. C. D. Nucleotides Nucleic Acids The 4 S’s Structure of the Information 1. 2. 3. E. Exceptions to flow Structure Levels of Control Recombinant DNA: Biochemical Basis of Biotechnology 1. 2. Restriction enzym es, DNA ligase Vectors and Inserts to m ake recom binant DNA (rDNA) 3. 4. 5. 6. F. Transformation of hosts Selection of transform ants Expression Site-directed m utagenesis Replication 1. Polym erases Lipids & Membranes A. Lipids 1. 2. a. b. c. d. e. 2. Fidelity a. b. c. d. Polymerase recognition Exonuclease Mis-match repair Post-replication repair 3. Sequence determination 4. PCR G. Transcription 1. 2. 3. 4. H. Overview-mRNA structure Process RNA Polymerase Fidelity 1. Genetic Code a. b. 1. 2. Structure Anti-codon Acylation (charging) i. Aminoacyl-tRNA Synthetases ii. Mechanism iii. Fidelity Protein Biosynthesis a. b. c. d. e. Overview Process Ribosome review Peptidyl transferase fidelity Introduction The 4 S’s a. b. c. d. 3. 4. 5. 6. Size Solubility Shape Stability Models for Membrane structure a. b. c. d. tRNA a. b. c. 3. Triplet Deciphering Fatty Acids Fats Waxes Membrane lipids Terpenes B. Membranes Translation 2. Roles Classes Old Model Data Fluid Mosaic Model Testing the model The Red-Blood Cell Membrane Membrane Asymmetry Membrane Fluidity Dysfunction in Ganglioside Recycling Leads to a Variety of Medical Disorders globoside 1 Lipids: Membranes The 4 S’s Size Shape Solubility Stability Lipids: Membranes Stability: Biochemical • Most cells continually degrade and replace their membrane lipids. • Phospholipids are degraded by phospholipases A−D. N. naja C. adamanteus 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 polar and/or 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 could 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