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BIOC 460, Summer 2010 Membrane Proteins Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 12, pp. 336-348 Bacteriorhodopsin Jmol structure: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/rhodopsin/rhodop1.htm PGH2 Synthase (COX 2) Jmol structure: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/cox12/cox121.htm Bacterial porin Jmol structure: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/porin/newporin.html Dynamics of membrane lipids as well as the proteins embedded with the lipid bilayer and is highly recommended: http://multimedia.mcb.harvard.edu/anim_innerlife.html Key Concepts • • • • • • Membrane Proteins Membrane functions (review): selective permeability barriers, information processing, organization of reaction sequences, energy conversion Lipids (lipid bilayer) responsible for permeability barrier Proteins perform essentially all other membrane functions, including modulation of permeability barrier by allowing or assisting some solutes to cross membrane (transport processes) Fluid mosaic model of membrane structure: 2-dimensional "fluid" composed of lipids and proteins (both often with attached carbohydrates on outer side of membrane) Proteins: peripheral, lipid-anchored, or integral Mobility of components within the membrane: – Lateral diffusion: rapid for both proteins and lipids within the plane of the membrane (except for proteins anchored, for example to cytoskeleton) – Transverse diffusion ("flip-flop") of both proteins and lipids is extremely slow, unless mediated by protein "flippases". – Lipid composition in the 2 leaflets of bilayer is asymmetric, as is protein distribution. 1 BIOC 460, Summer 2010 Key Concepts, continued • • • Membrane fluidity essential to function, regulated by fatty acid composition of lipids and (eukaryotes) by cholesterol content Integral membrane proteins typically assume one of two secondary structures to get polar groups of polypeptide backbone across hydrophobic core of lipid bilayer: membrane-spanning 〈 helices (about 20 residues long) or antiparallel ® sheets wrapped into ® barrels. Examples: – Glycophorin: single hydrophobic transmembrane 〈 helix – Bacteriorhodopsin: 7 transmembrane helices – Prostaglandin H2 Synthase: on surface of ER membrane but anchored in membrane by a set of 〈-helices with hydrophobic R groups that extend into membrane core – Porins: large ® barrel with aqueous channel down the center Membrane Proteins • mediate nearly all membrane functions except establishment of permeability barrier • membrane protein functions: – "pumps" (active transport) – "gates" ("passive" transport, facilitated diffusion) – receptors – signal i l ttransduction d ti – enzymes – energy transduction • Membrane protein distribution: – Both amount of protein in general and which specific proteins are present varies with function of membrane, i.e., with type of membrane and with cell type. – Examples: • myelin (membrane around myelinated nerve fibers, function=electrical insulation) mostly lipid (only ~18% protein) • plasma membrane: enzymes, receptors, etc. (~50% protein) • mitochondrial inner membrane and chloroplast thylakoid membrane: electron transport, energy transduction (ATP synthesis) (~75% protein) Membrane Proteins 2 BIOC 460, Summer 2010 Schematic Diagram of Membrane Structure Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 11-3 Fluid Mosaic Model of Membrane Structure (Singer and Nicholson, 1972) • A 2-dimensional "solution" of globular proteins embedded in fluid lipid bilayer, structurally and functionally asymmetric with respect to the 2 sides of bilayer • Proteins and lipids: free lateral diffusion (in plane of membrane). • Transverse diffusion ("flip-flop") is very slow (thermodynamic and kinetic barrier) without a catalyst catalyst. • Carbohydrates on both proteins (glycoproteins) and lipids (glycolipids) are exposed on extracellular surface of plasma membrane. Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 11-3 Membrane Proteins 3 BIOC 460, Summer 2010 Membrane Asymmetry: Lipids • • • • • Biological membranes are asymmetric with respect to lipids Lipid asymmetry is related to function: Plasma membrane: PS required in outer leaflet for platelet formation of blood clot Other cells: PS in outer leaflet signals for apoptosis. Cardiolipin: (not shown) only found in mitochondrial membranes Membrane Asymmetry: Proteins • • • • • Membrane Proteins Asymmetric orientation of proteins in planar membrane. Protein orientation: Proteins definitely oriented: inner side, or outer side side, or spanning membrane, but in a specific orientation Orientation established during proteins synthesis in ER Orientation/structure DIRECTLY linked to function: cell recognition, signaling into cell, transport of ions (H+, Na+, K+, sugars) in or out of cell. Oft structures Often t t can be b quite it elaborate: the mitochondrial cytochrome bc1 complex. 4 BIOC 460, Summer 2010 Membrane Proteins 3 types based on association with membrane: 1.Peripheral 2.Integral 3.Lipid-anchored 1. Peripheral membrane proteins • weakly associated with membrane surface • bind to polar lipid heads and/or to integral membrane proteins –electrostatic interactions (ionic bonds and/or hydrogen bonds) –easily extractable from membranes by high salt concentrations (disrupting electrostatic interactions), or by EDTA (chelates Ca2+ and Mg2+) • usually water-soluble • Can also be fibrous proteins attached to membrane surface (cytoskeletal proteins). 2. Integral membrane proteins • tightly bound to membrane - interact with interior (membrane core, lipid tails) (hydrophobic interactions) • require detergents (or organic solvents) for extraction • water-insoluble • both peripheral and integral domains. • can completely span membrane ("transmembrane proteins"). • Glycoproteins always have carbohydrates on extracellular side side. – carbohydrates attached by O-glycosidic bonds to OH groups of Ser or Thr or – N-glycosidic bonds to Asn Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 11-3 Membrane Proteins 5 BIOC 460, Summer 2010 3.Lipid-anchored membrane proteins • Covalently linked to lipids, required for association with membrane • Can be reversibly attached to/detached from proteins. – "switching device" to alter affinity of protein for membrane – e.g., N-myristoylation: C14 fatty acid in amide linkage to N N-terminal terminal Gly examples: –PKA (cAMPdependent protein kinase) −α subunit of G proteins Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 11-3 Transmembrane Proteins Hydrogen Bonding: an extremely important consideration for inserting the polar backbone of a protein into a lipid membrane! • By hydrogen-bonding all polar backbone groups via formation of secondary structures, α-helical or β barrel motifs for membranespanning portions of transmembrane protein protein, unfavorable thermodynamic conditions are minimized. Examples of Transmembrane Proteins demonstrating transmembrane secondary structural motifs: 1. glycophorin A (erythrocyte membranes) 2. bacteriorhodopsin (purple membrane of Halobacterium halobium, a saltloving bacterium) 3. prostaglandin H2 synthase (COX, enzyme involved in biosynthesis of prostaglandins/inflammatory response) 4. porins (channel-forming proteins -- outer membranes of gram-negative bacteria and outer membranes of mitochondria and chloroplasts) Membrane Proteins 6 BIOC 460, Summer 2010 1. Glycophorin A: • A glycoprotein: by mass ~60% carbohydrate, ~40% protein. • Single transmembrane α-helix • Most of protein (N-terminal portion) on outside of cell, exposed to water; mainly hydrophilic residues, heavily glycosylated (carbohydrates in glycosidic bonds to Ser, Thr, and Asn) • Carbohydrates: ABO and MN blood group antigen-determining structures. • Extracellular part of protein also receptor for influenza virus binding to cells • C-terminal portion on cytosolic side of membrane, interacts with cytoskeletal proteins 19-AA AA residue hydrophobic segment is exactly right length to span • One 19 membrane if it’s coiled into an α-helix -- hydrophobic R groups oriented outward, toward "solvent" (hydrophobic core of lipid bilayer). • Hydrophobic 19-20 amino acid α-helices are very common way for proteins to span biological membranes. • Polar peptide backbone groups (carbonyl oxygens and amide N-H groups) fully hydrogen-bonded – Hydrogen-bonding "neutralizes" these polar groups, and – "screens" them from contact with lipid core by R groups on outside of helix. 30 Å across, across so 20-residue 20 residue α α-helix helix (20 • hydrophobic core of membrane is ~30 residues x 1.5 Å "rise" per residue) is right length to reach across hydrophobic core. Berg et al., Fig. 12-27a Membrane Proteins 7 BIOC 460, Summer 2010 2. Bacteriorhodopsin • from purple membranes of bacteria of the genus Halobacterium, salt-loving archaebacteria • 7 transmembrane α-helices (common motif for signaling proteins: "7-TM helix receptors" • 247 amino acid residues (26,000 MW) with retinal cofactor (absorbs light) • uses light energy to pump protons across membrane, out of cell, against a concentration gradient (example of primary active transport) • generates and maintains [H+] gradient (pH gradient) across cell membrane • transmembrane proton gradient = "stored" potential energy, used by ATP synthase to drive ATP synthesis Berg et al., Fig. 12-18 •globular shape, most of protein embedded in membrane b •retinal (not shown) in middle of bundle http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/rhodopsin/rhodop1.htm 2. Bacteriorhodopsin • 7 TM helices closely packed; at 10o angle to plane of membrane • outer sides of helices (interact with hydrophobic interior of membrane): many hydrophobic R groups • "inner" sides of helices have some charged/polar residues to interact with retinal and for proton translocation • Opposite of water-soluble globular proteins: in an integral membrane protein not only are most R groups on interior of protein hydrophobic protein, hydrophobic, but R groups on outside of protein are hydrophobic as well Berg et al., Fig. 12-18 Membrane Proteins 8 BIOC 460, Summer 2010 3. Prostaglandin H2 synthase • COX, enzyme involved in biosynthesis of prostaglandins/inflammatory response • inhibited by aspirin and other NSAIDs • integral membrane protein, homodimeric, with subunit structures primarily α-helical • does NOT span membrane -- it's on surface of endoplasmic reticulum (ER) membrane extending into lumen of ER membrane, • firmly anchored in membrane by α-helices with hydrophobic R groups Berg et al., Fig. 12-23 Berg et al., Fig. 12-24 3. PGH2 synthase • Localization important to binding substrate, arachidonic acid, released from membrane lipids by PLA2. • Substrate can enter enzyme active site via hydrophobic channel (faces membrane) without entering aqueous solvent. • NSAIDs block channel, inhibit enzyme, prevent prostaglandin synthesis, and reduce inflammation. (What type of inhibitor would naproxen be -competitive, titi noncompetitive, titi or uncompetitive? titi ? What Wh t is i the th mechanism of Aspirins inhibitions of COX?) Berg et al., Fig. 12-23 Membrane Proteins Berg et al., Fig. 12-24 9 BIOC 460, Summer 2010 4. Porins: a β−barrel for H2O transport • channel-forming proteins in outer membranes of gram-negative bacteria, mitochondria, and chloroplasts • antiparallel β−barrel, with hydrophobic R groups facing membrane R-groups membrane, polar R Rgroups facing interior of barrel, stabilizing H2O. What is significance of β-sheet with respect to peptide backbone carbonyl O and amide N-H groups? Berg et al al., Fig Fig. 12 12-20 20 Jmol routine: http://www.biochem.arizona.edu/classes/bioc462/462a /jmol/porin/newporin.html Berg et al., Fig. 2-50 Lipid Rafts or Microdomains • In late 1990’s a modification of the Fluid-Mosaic Model emerged • On long timescales (msec – sec) lipids diffuse freely • On shorter times (µsec) lipids appear pp localize,, then “hop” p to another region • Phosphosphingolipids and cholesterol seem to cluster together in “rafts” • ~50 nm diameter; significantly thicker • Few thousand lipids; 10 – 50 proteins • Specificity S ifi it ffor ttype off proteins t i localized • Organize receptors or signaling proteins(?) Membrane Proteins 10 BIOC 460, Summer 2010 Rafting in the Sea of Lipid Rafts can be visualized by Atomic Force Microscopy (left). The cantilever acts like the stylus arm on an “ancient” record player. Th peaks The k b below l are GPI GPI-linked li k d proteins protruding above raft (green). Rest of membrane (black). Learning Objectives • • • • • Membrane Proteins Terminology (as applied to membrane proteins): peripheral, integral, lipid-anchored (operational definition -- how can they be extracted from membrane?); trans-membrane helix; antiparallel β barrel Briefly explain what “lipid rafts” are in membranes. What are primary lipid components? Explain in structural terms how an integral membrane protein can deal with its polar backbone groups in spanning the hydrophobic core of a lipid bilayer. Name 2 types of secondary structural elements used by integral membrane proteins to cross membranes. Describe where the R groups are located in these secondary structural elements relative to the hydrophobic lipid core. Discuss the structural properties of the following examples of membrane proteins: glycophorin A, bacteriorhodopsin, prostaglandin H2 synthase, and a porin. Include in your discussion the type(s) of secondary structure and types of R groups found in the transmembrane (membrane-spanning) structural components of these membrane proteins. 11