9 1 MCB 450 Lecture 9 Membrane Proteins peripheral integral lipid anchored Membrane Transport 9 2 Fluid mosaic model for membrane organization 9 3 Membranes are full of proteins Membranes provide a 2 D scaffold for membrane associated proteins Membranes can be 20 80 protein dry wt In eukaryotes different membranes can differ in their protein composition Proteins can be associated with membranes in 3 different ways Demonstration identification of membrane proteins Isolate membranes e g by density gradient centrifugation Detect proteins in membrane fraction e g following solubilization of membranes in SDS mercaptoethanol and separation by SDS PAGE 9 4 Association of proteins with membranes 1 Membrane proteins are distinguished operationally by the conditions required to release them 1 Integral removable only by agents that interfere with hydrophobic interactions i e detergents proteins that pass through membane at least once Most membrane proteins fall into this category 1 3 of the different proteins in most cells INSIDE CELL 9 5 Association of proteins with membranes 2 2 Peripheral released by relatively mild treatments that interfere w electrostatic interactions or break H bonds changes in ionic strength or pH removal of Ca2 by chelator urea low conc of mild detergent 3 Lipid linked some proteins released by treatment with phospholipase C cleaves a glycosylphosphatidylinositol GPI membrane anchor attached at COOH terminus of some proteins other types of protein lipidation too OUTSIDE CELL 9 6 Integral membrane proteins 1 1 Attachment of an integral protein to a membrane requires protein has one or more hydrophobic domains that can span the lipid bilayer Most often these are helical domains but barrel membrane proteins possible 2 A given transmembrane protein always has the same orientation in the membrane Example glycophorin a protein from the red cell membrane i extracellular domain with covalently attached sugars ii stretch of hydrophobic buried in core of membrane iii C terminal domain in cytoplasm 9 7 Integral membrane proteins 2 Example adrenergic receptor in plasma membrane i N terminal extracellular domain with covalently attached sugars ii 7 stretches of hydrophobic buried in core of membrane iii hydrophilic loops of varying length between hydrophobic segments iv C terminal domain in cytoplasm 9 8 Transmembrane domains properties 1 1 Transmembrane domains are usually helical segments of with non polar side chains Segments long enough to span the bilayer 2 Why an helix Internal H bonding between polar CO NH groups of peptide bonds in helix lowers energetic cost of transferring them into hydrocarbon interior Also the non polar R groups of the in the chain make hydrophobic interactions w surrounding lipids which further stabilizes the helix helix formation favored when chain surrounded by lipids and there are no H2O molecules w which to form H bonds 9 9 Transmembrane domains properties 2 3 Length of a helix 1 5 per width of hydrophobic core of bilayer 30 so a helix of 20 25 would span the thickness of the bilayer X ray structure of bacteriorhodopsin 7 transmembrane helices 20 hydrophobic clustered together space around and between filled by membrane lipids 9 10 A particular class of may be enriched on one side of an helix 100 Remember from Lect 3 NON POLAR 100 Each is related to next by a rise of 1 5 and a rotation of 100 so 3 6 per 360 turn rising at 1 5 per 100 In an amphipathic helix polar residues are spaced in the 1 sequence such that they end up distributed on one face of the helix with non polar on the other side 9 11 Amphipathic helices often form bundles often seen in transport proteins Polar faces of helices oriented towards a central cavity non polar faces oriented towards hydrophobic lipid core Produces a transmembrane channel lined with polar provides many opportunities for H bonding with solute moving through e g glucose 9 12 Transmembrane segments can be predicted hydropathy plots 1 Give each side chain a score according to its relative hydrophobicity Phe gets high ve score Arg a low ve score 2 Sum the hydrophobicity score for successive segments of peptide windows of 11 20 e g s 1 15 2 16 3 17 etc hydropathy index 3 Plot hydropathy index vs residue number of 1st in window 9 13 Hydropathy plots identify helical transmembrane segments 4 A stretch of 20 with a high hydropathy index presumed to be an helical transmembrane segment a k a membrane spanning domain Typically L I V M F A W Y at membrane aqueous interface HYDROPHOBIC HYDROPHILIC Kent and Robertson BMC Evolutionary Biology 2009 9 41 9 14 Examples of hydropathy plots COVALENTLY ATTACHED CARBOHYDRATES NOT INCLUDED IN THE ANALYSIS ABOVE 1 5 IN THIS ANALYSIS 1 TM domain 7 TM domains 9 15 barrel membrane proteins sheets curl up to form hollow cylinder which can serve as a pore Non polar side chains face membrane interior 9 16 barrel membrane proteins are usually not identifiable using hydropathy analyses for membrane spanning helices not easy to identify from sequence Hydropathy plot http sbcb bioch ox ac uk bond php 9 17 Solubilization of integral membrane proteins principle REPLACE LIPIDS W DETERGENTS 9 18 Solubilization of integral membrane proteins different detergents can be used 1 Strong ionic detergents such as SDS unfold the entire protein by binding to internal core SDS often denatures proteins NON POLAR NON POLAR 2 Non ionic detergents such as Triton X 100 bind mainly to the membrane spanning segments of proteins can allow active protein to be recovered MORE POLAR POLAR polyoxyethylene octyl phenyl ether 9 19 It s harder to get 3 D structures of membrane proteins Hard to crystallize Have both hydrophilic and hydrophobic regions Not soluble in aqueous buffer solutions but traditional crystallization techniques use aqueous solvents Sometimes certain detergents can be used to solubilize a membrane protein in its native conformation get crystals of protein detergent complex very hard to find the right conditions 9 20 Lipid anchored proteins Thioester linked 16 0 palmitate Amide linked 14 0 myristate at N terminus CYTOPLASMIC FACE OF MEMBRANES Only palmitoylation is reversible Thioether linked 15 or 20 carbon isoprenoid on C terminal Cys C15 farnesyl C20 geranylgeranyl The lipid anchors are added after a protein has been made cytoplasmic side extracytoplasmic 9 21 Lipid anchored proteins EXTRACYTOPLASMIC FACE OH HO HO OH O O O HO HO H N N H OH O O OH HO HO O O P R O O O HO HO OH O OH O O HO OH NH 3 O O
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