Class Note for BIOC 460 at UA 5
Class Note for BIOC 460 at UA 5
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Date Created: 02/06/15
BIOC 460 Spring 2008 Lecture 19 Membranes 2 Membrane Proteins Next 2 lectures Membrane Transport Reading Berg Tymoczllto amp Stryer 6th ed Chapter 12 pp 336348 Bacteriorhodopsin Jmoi structure nttg vavvblulnerrl arlzuna eduEiasseSbluE4EZ4B23 muirnudugslnrnudugi ntrn PGHZ Synthase cox 2 Jmoi structure i 4 i Bacterial porin Jmoi structure imp vWWlblulnern allzurla eduEiasseSbluE4EZ4B23 muigurlnnemurln iltrni Key Concepts Membrane functions reyiew selectiye permeability barriers information processing organization of reaction sequences energy conyersion 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 Zedlmerlslorlai fluid composed of lipids and proteins both often with attached carbohydrates on outer side of membrane Proteins peripheral lipidanchored or integral Mobility of components within the membrane 7 Lateral diffusion rapid for both proteins and lipids within the plane ofthe membrane except for proteins anchored for example to cytoskeietorl 7 Transverse diffusion flipflop of both proteins and lipids is extremely slow unless mediated by protein flippases 7 Li id composition in the 2 leaflets of bilayer is asymmetric as is protein distribution Key Concepts continued Membrane fluidity essential to function regulated by fatty acid composition of lipids and euxaryotes by cholesterol content integral membrane proteins typically assume one of two secondary structures to get polar groups of polypeptide bacxbone across hydrophobic core of lipid bilayer membranespanmng a helices about 20 residues long or antiparallel 3 sheets wrapped into 3 barrels Examples 7 Glycophorin single hydrophobic transmembrane ahelix e Bacteriorhodopsin 7 transmembrane helices e Prostaglandin H2 Synthase on surface of ER membrane but anchored in membrane by a set of aeneilces with hydrophobic R groups that extend into membrane core 7 Porins large 3 barrel with aqueous channel down the center Learning Objectives Terminology as applied to membrane proteins peripheral integral llpldeanchnred operational definition 77 how can they be extracted from membrane transemembrane helix antiparallel 5 panel Briefly explain what lipid rafts are in membranes Explain in structural terms how an integral membrane protein can deal with its polar bacxbone groups in spanning the hydrophobic core of a lipid bilayer Name 2types of secondary structural elements used by integral membrane proteinsto cross membranes Describe where the R groups are located in these secondary structural elements relatiye to the hydrophobic lipid core Discussthe structural properties of thefollowing examples of membrane proteins glycophorin A bacterlornodopslrl prostaglandin H2 synthase and a porin include in your discussion thetypes of secondary structure and types of R groups found in the transmembrane membranespanmng structural components of these membrane proteins Lipid Rafts lateral diffusion sideways in plane of membrane rapid for lipids and for many proteins but NOT for proteins anchored by attachment to other proteins e gin cytosxeleton on inner side of membrane or extracellular matrix or both 77 e g flbrorlectlrl Timescale of msec to sec lipids DO diffuse freely in plane of membrane 0n much shortertime scale 25 psec intervals lipids zoom around in a small confined area of membrane then seem to hop to another small confined area as if there were fences they had to hop oyer networks of interacting membrane protelrls yery recent research different types of membrane lipids are NOT randomly located all through the membrane 77 some phosphosphingolipids and cholesterol cluster together in membrane rafts patches of lipid about 50 nm wide thicker and more ordered structurally than the more disordered neighboring microdomains rich in glycerophospholipids Lipid rafts haye afew thousand lipid molecules and about 10750 protein molecules and are particularly enriched in 2 classes of ilpldearlcnored integral membrane proteins Rafts may organize some membrane receptors and signaling proteins whose functions require protelrleprotelrl interactions collision probability much greater in corrai of local raft Proteins and lipids moye in and out of rafts but on slower time scale sec rather than psec but biochemical processes work on thefaster time scale in which rafts are stable clusters Membrane Proteins mediate nearly all membrane functions except establishment of permeability barrier membrane protein functions 7 pumps actiye transport 7 gates passiye transport facilitated diffusion 7 receptors 7 signal transduction e enzymes 7 energy transduction Membrane protein distribution 7 Both amount of protein in general and which speci c proteins are presentyanes with function of membrane i e with type pf memprane and with cell type 7 Examples myelin membrane around myelinated nerye fibers functionelectrlcal insulation mostly lipid only i8 protein plasma membrane enzymes receptors etc 50 protein mitochondrial inner membrane and chloroplast thylakoid membrane electron transport energy transduction ATP synthesis 75 protein LEC 19 Membranes 2 Membrane Proteins BIOC 460 Spring 2008 Fluid Mosaic Model of Membrane Structure Schematic Diagram of Membrane Structure Singer and Nicholson 1972 on on mide model of biological membranes as 2dimensional quotsolutionsquot of 9 h chainsol globular proteins embedded In fluld lipid bilayer structurally and V glycoprolein functionally asymmetric with respect to the 2 sides of bilayer Fatty acyl chains in interior of membrane form a fluid hydrophobic region surface is hydrophilic Proteins and lipids can diffuse laterally in plane of membrane unless they39re quotanchoredquot to something Dligosaccharide O C 2 n m 1 1 but transverse diffusion quotflipflopquot is very quot39 Elixgmn l slow unless there39s a catalyst for it s 39 erquot I 4 l Carbohydrates quot Jamalquot I k on both proteins w39mm x3 ou s39de ii p mu m glycoproteins a l I ah 1 z y WIN ViiWm l l and lipids I a 0 4 4 f a quot f warheads 57 A Inside 1 glycollp39ds 1 F39 39 WC 397 393 r l39 m Lipid p Slerol l are exposed on Illlfwui INN Milt gt V bllayer LL In extracellular qlllgllllllmlllg rial illiii lillu Ml 7 39 l 39 quot surface of Phasphol Wirequot 3 I quotFegl39alpmmquot Paint 239 integral protein Plasma Wm d5 9 Steml Inquot 9 5 b P h slnge vans pro eln 7 A 351 membranehelix Solalderlllly d agging membrane rib I a C1 i 39quot quotquot Nelsomwwge39 p 2 39quot 52 PHW peSOfB Whem SW 351513 mergbnnehelix covalently m g a f i39m Nelson amp Cox LehnlngePlinclpes ofBochemisly 4th ed Flg 1173 4th ed Fig 1173 linked lolipid Membrane Proteins Membrane Protein Asymmetry 3 types based on association with membrane All biological membranes are asymmetric 1 Peripheral Membrane protein orientation Proteins definitely oriented inner side 2 Integral or outer side or spanning membrane but in a specific orientation 3 Lipidanch red Example asymmetry of the NaK transport system in plasma 39 membranes 1 Peripheral membrane proteins Transport enzyme pumps Na out ofceII and K into cell against weakly associated with membrane at sunace a concentratiqn gradient unfavorable S9 reqUireS quotmut 0f free bind to polar lipid heads andor to integral membrane proteins energy coupling to processes resulting in net hydrolysrs of ATP on inside of cell electrostatic interactions predominate ionic bonds andor Plasma membrane hydrogen bonds ofcell easily extractable from membranes by high salt concentrations disrupting electrostatic interactions or by EDTA chelates Ca and Mg usually watersoluble globular There can also be fibrous proteins attached to membrane surface cytoskeletal proteins Some peripheral quotmembranequot proteins come and go from membrane eg using reversibly attached lipid anchor K Covalently attached C14 fatty acyl chain myristoyl group slips Berg etaIFig1234 into lipid bilayer holding protein to surface of membrane 3 Lipidanchored membrane proteins 239 39Ptegra39 membrane pr te39ns Proteins that are covalently linked lipids a requirement fortheir 39 gQIIlS lghsgrggh gb emgfggaggrad Wlth Interlor membrane Gore llpld assomation With the membrane lipid inserts into membrane assocrating protein with membrane 39 gigg39 nii irgzms or orgamc SOIVentS for eXtraCt39on from membranes Some lipid anchors can be reversibly attached toldetached from I 39 proteins 32m Eg ilgie i iggtn gni g agnigi i ilrgggggf in8quot quotswitching devicequot to alter af nity of protein for membrane 39 role in signal transduction pathways in eukaryotic cells Glycoprotems always have carbohydrates on extracellular Sidegr h yd e gquot Nmy qyla on CHM fatty acid attached migmmme carbohydrates attached by OglyCOSIdIC kg1ng 3quot enzymatically in amide linkage to aamino of l hainsol I acetal bonds to OH groups of specific 39 397 17 nlymprotein Nterminal Gly of membrane protein 39l E39V jp 39quot Ser or Thr residues or examples of reversibly L Nglycosidic GlyLollpid FR ou39side l 7 Sgggi sstoylated Glyltalipid Omside bonds to Asn M Q V residues 4 f z 3 3 Q j PKACAMP 39 a z a z a u fl 7 M deptendent g N gt 39 aw quot c l M pro eln 5 4 up N h tlllll ll Il39Ianllll in m gLKlu kinase mszm l Rh39 iunmgm my lijll lul Il lli NM 1 3 gsubtur1it of 4 9 all Millyll ll rwml l gt l 107 l s inside R 1 P ems 13mm Tn l A inside 7 7 7 Skevnl f J l L Steml I V p0 o e gtJ 1 80 0 Nelson amp Cox Lehnlnger M hm l lg eLgfgiquot may integgzlpmln Nelson amp Cox Lem we M Ml rsgslghv ffin I D25mg inlegalpmmln hlngfle gr oschemlslry prglein membrane helix n l pid m g i cm f zf e gof oghemSVY Stein membrane helix awash m f zf ga i39m LEC 19 Membranes 2 Membrane Proteins BIOC 460 Spring 2008 How does an integral membrane protein accommodate its polar really ALL of them ie by forming secondary structures either ahelical or 3 barrel motifs for membranespanning parts of transmembrane protein Examples of Transmembrane Proteins demonstrating Transmembrane Proteins backbone peptide NH and 00 groups in a stable way across hydrophobic core of lipid bilayer By hydrogenbonding as many as possible of polar backbone groups transmembrane secondary structural motifs glycophorin A erythrocyte membranes 2 bacteriorhodopsin purple membrane of Halobacterium halobium a salt loving bacterium 3 prostaglandin H2 synthase COX enzyme involved in biosynthesis of prostaglandinsinflammatory response 4 porins channelforming proteins outer membranes of gramnegative bacteria and outer membranes of mitochondria and chloroplasts Hydrophobic 1920 amino acid sequences are a ver common way for proteins to span biological membranes in orhelica conformation Polar peptide backbone groups carbonyl oxygens and amide NH groups fully hydrogenbonded Hydrogenbonding quotneutralizesquot these polar groups and quotscreensquot them from contact with lipid core by R groups on outside of helix hydrophobic core of membrane 2X length of tails is 30 A across so 20 residue ccheliX 20 residues x 1 5 A quotrisequot per reSIdue is right length to reach across hydrophobic core Outside Bilayer Inside Berg et al Fig 1227a a 2 Bacteriorhodopsin Most of protein structure in 7 closely packed transmembrane helices arranged in a bundle almost perpendicular to plane of membrane outer sides of helices interact with hydrophobic interior of membrane many hydrophobic R groups quotinnerquot sides of helices facing interior of bundle have some charged residues some chargedpolar residues interact with cofactor retinal some involved in proton translocation Arrangement sort of opposite of watersoluble globular proteins in an integral membrane protein not only are most R groups on interior of protein hydrophobic but R groups on outside of protein are hydrophobic in contact with W membrane lipid core Can have hydrophilic groups in interior if needed to interact with prosthetic group like retinal or with water in channels as in porin structures Berg et al Fig 1218 LEC 19 Membranes 2 Membrane Proteins 1 Glycophorin A has a single transmembrane ccheliX integral membrane protein in erythrocytes total MW 31000 but only 131 aa residues Remember average quotresidue massquot of an amino acid residue in a protein is about 110 so glycophorin A would have MW about 14400 Where does additional mass come from Glycophorin A is a glycoprotein by mass 60 carbohydrate 40 protein Most of protein Nterminal portion on outside of cell exposed to water mainly hydrophilic residues heavily glycosylated lots of carbohydrates in glycosidic bonds to specific Ser Thr and Asn residues Carbohydrates include ABO and MN blood group antigendetermining structures Extracellular part of protein also receptor for influenza virus binding to cells Cterminal portion on cytosolic side of membrane interacts with cytoskeletal proteins One 19AA residue hydrophobic segment is exactly right length to span membrane if it s coiled into an ahelix with hydrophobic R groups oriented outward toward quotsolventquot hydrophobic core of lipid bilayer 2 Bacteriorhodopsin 7 transmembrane ahelices common motif for integral membrane proteins involved in signaling processes quot7TM helix receptorsquot from purple membranes of bacteria of the genus Halobacterium saltloving archaebacteria 247 amino acid residues 26000 MW When oxygen is scarce uses light energy to pump protons across membrane out of cell against a concentration gradient example of primary active transport generates and maintains Ht gradient pH gradient across cell membrane Resulting transmembrane proton gradient quotstoredquot potential energy used by a different protein ATP synthase to drive ATP synthesis in a photosynthetic mode globular shape most of protein embedded in membrane Jmol structure of bacteriorhodopsin Berg et al Fig 1218 3 Prostaglandin H2 synthase COX enzyme involved in biosynthesis of prostaglandinsinflammatory response cyclooxygenase COX activity inhibited by aspirin and other nonsteroidal antiinflammatory drugs NSA Ds like ibuprofen naproxen Celebrex Vioxx etc intreglral membrane protein homodimeric with subunit structures primarily cc e ica protein does NOT span membrane it39s on surface of endoplasmic reticulum ER membrane extending into lumen of ER but firmly anchored in membrane by a set of ahelices with hydrophobic R groups extending into membrane core Hydrophnhkamlnn add side chains rxx llll llll c Ser 520 5 Berg et al Fig 1224 Berg et al Fig 1223 BIOC 460 Spring 2008 3 PGH2 synthase REV EW the notes on enzyme inhibition and examples of irreversible and reversible inhibitors and lipids notes on phospholipases substrate arachidonic acid is a 20C fatty acid VERY hydrophobic released from membrane lipids by PLAZ Substrate can enter enzyme active site via hydrophobic channel in the enzyme without entering aqueous environment NSA Ds block that channel inhibiting the enzyme preventing prostaglandin synthesis and thus reducing inflammation What type of inhibitor would naproxen be competitive noncompetitive or uncompetitive Jmol structure of COX cyclooxygenase with inhibitor in substrate entry channel Aspirin acetvlates Ser 530 residue in channel Hydrophabkamlno D acid side chains r nixxxxxirx up c ugn A 9 Hydrophobic channel Berg et al Berg et al Fig 1223 Fig 1224 Set 530 4 Porins This is a review from Lecture 6 so we will not go over it again in class but it39s part of the Lecture 19 material channelforming proteins found in outer membranes of gramnegative bacteria and also in outer membranes of mitochondria and chloroplasts trimers of 3 identical subunits in the membrane homotrimers each subunit a large 16 or 18stranded antiparallel Bsheet rolled up into a large 3 barrel Remember R groups in 5 sheets alternate quotsticking outquot on opposite sides of the sheet Polar R groups line aqueous central channel across membrane and also for residues interacting with H20 at surfaces of membrane Hydrophobic R groups face outside of barrel in contact either with other subunits or with hydrophobic core of membrane Polar peptide backbone groups carbonyl O and amide NH groups fully hydrogenbonded in B barrel secondary structure Water lled largely hydrophobic L Berg et al Fig 2 50 Structure of one subunit of a bacterial porin Jmol structure of a orin subunit left side view in plane of membrane right view from periplasmic space from inside looking out through pore in outer membrane i Berg et al Fig 1220 On a single strand in B conformation where are the side chains of the amino acid residues all on one side Alternating sides 3 residues R groups on one side 1 on the other side Amino acid sequence of a porin 5 strands are indicated with diagonal lines indicating direction of hydrogen bonding along the 5 sheet hydrophobic residues F l L M V W and Y shown in yellow Berg et al Fig 1221 Note the more or less alternating hydrophobic and hydrophilic residues in the B strands adjacent R groups project out from sheet on opposite sides LEC 19 Membranes 2 Membrane Proteins
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