Chapter7– Biomembrane Structure

Whatwillyoulearn?

Biomembranestructure

Transmembranetrasportofionsandsmallmolecules

Movingproteinsintomembranesandorganelles

Vesiculartraffic,secretionandendocytosis

SignaltransductionandGprotein-coupledreceptors1

SignaltransductionandGprotein-coupledreceptors2

Signalingpathwaysthatcontrolgeneactivity

Eukaryoticcellcycle

CancerandImmunoolgy

Cellularsignaling…

Chapter7– Biomembrane Structure

7.1TheLipidBilayer:CompositionandStructuralOrganization7.2MembraneProteins:StructureandBasicFunctions7.3Phospholipids,Sphingolipids,andCholesterol:SynthesisandIntracellularMovement

7.1TheLipidBilayer:CompositionandStructuralOrganization• Membranes̶barriersbetweenaqueouscompartments• Amphipathicphospholipidsspontaneouslyformbilayerswithhydrophilicfacesandahydrophobiccore.

• Biologicalmembranes– varyinlipidcomposition– impermeabletowater-solublemoleculesandions– haveaviscousconsistencywithfluid-likeproperties

Plasmamembrane?What’stheroleofplasmamembrane?

Fluidmosaicmodelofbiomembranes

Membraneandmembraneproteinlikethis…

Fluidmosaicmodelofbiomembranes

• Membrane:phospholipidbilayer– 2Dfluid• Fluidity:(similartothatofoliveoil)

• Integralmembraneproteins(transmembraneproteins)spanthebilayerandoftenformdimersandhigher-orderoligomers.

• Lipid-anchoredproteins– tetheredtooneleafletbyacovalentlyattachedhydrocarbonchain• Peripheralproteins– associateprimarilybyspecificnoncovalentinteractionswithintegralmembraneproteinsormembranelipids[includecytoskeletalproteins]

Fluidmosaicmodel

https://www.youtube.com/watch?v=Qqsf_UJcfBc

Eukaryoticcellularmembranesaredynamic structures.

• (a)HIV-infectedcellplasmamembrane:• Viruscore– envelopedbyregionofcellplasmamembranethatcontainsspecificviralproteins• HIVparticles– budfromplasmamembrane

• (b)StackedmembranesoftheGolgicomplexwithbuddingvesiclesinvolvedinintracellulartrafficking

Thebilayerstructureofbiomembranes.

•Membranephospholipids:• Amphipathicmolecules–endshavedifferentchemicalproperties:• Hydrophobic (“waterfearing”)fattyacid-based(fattyacyl)hydrocarbon“tail”– partitionsawayfromwater• Hydrophilic (“waterloving”)polar“headgroup”– interactswithwatermolecules

Howtoextractthephospholipid?

Formationandstudyofpurephospholipidbilayers.

• Isolationandreconstitutionofmembranebilayers:• (Top):Membranetreatmentwithanorganicsolvent(3:1mixtureofchloroformandmethanol)selectivelysolubilizesthephospholipidsandcholesterol.(Proteinsandcarbohydratesremaininaninsolubleresidue.)• Solventremovedbyevaporation.• (Bottomleft):Mechanicaldispersalofextractinwater– lipidsspontaneouslyformliposomes• (Bottomright):Planarbilayerformationoverasmallholeinapartitionseparatingtwoaqueousphases– canbeusedtostudythephysicalpropertiesofbilayers,suchastheirpermeabilitytosolutes.

Thefacesofcellularmembranes

Whereisthemembrane?Function?Dynamics?

Thefacesofcellularmembranesareconservedduringmembranebuddingandfusion

Thenhowtoinserttheproteinintheplasmamembrane?

Variationinbiomembranes indifferentcelltypes

• (a)Erythrocytecell:• Discoid– flexibleshaperequiredforsqueezingthroughbloodcapillarieswithsmallerdiameters• Shapedefectscausecelllysis– anemias

• (b)Ciliummembrane(Ependymalcellsliningbrainventricles):• Regionofplasmamembranethatprojectsfromcell• Flexibilityenablesciliarymotility

Question

• Membranedynamics?• Howtomakethedifferentcellmembranes?

• Protein-membranetrafficking• Cellmovement• Membrane-supportingproteins(structuralproteins)

Basicstructureandproperty

Threeclassesofmembranelipids• (a)Phosphoglycerides (mostabundant):

• Glycerol backbone.• Tails– twoesterifiedhydrophobicfattyacylchains

• varyinlength(commonly16or18C)• varyinsaturation– saturated(nodouble

bonds)orunsaturated(one,two,orthreedoublebonds)

• Head– apolargroupesterifiedtothephosphate• 4majorheadgroups– phosphatidylcholine

(PC),phosphatidylethanolamine(PE),phosphatidylserine(PS),andphosphatidylinositol(PI).

• (Plasmalogens – onefattyacylchainattachedtoglycerolbyanesterlinkageandoneattachedbyanetherlinkageandthesameheadgroupsasotherphosphoglycerides)

• (b)Sphingolipids:• Derivativesofsphingosine(anaminoalcoholwitha

longhydrocarbonchain)• Variousfattyacylchainsconnectedbyanamidebond• Sphingomyelins(SM)– containaphosphocholine head

group• Somesphingolipidsareglycolipidsthatcontaina

singlesugarresidueorbranchedoligosaccharideattachedtothesphingosinebackbone.Glucosylcerebroside (GlcCer)hasaglucoseheadgroup.

• (c)Sterols:• Membranecomponents– animals(cholesterol),fungi

(ergosterol),andplants(stigmasterol)• Amphipathicstructure:

• headgroup– singlepolarhydroxyl• tail– conjugatedfour-ringhydrocarbon and

shorthydrocarbonchain

• Phospholipidswithlongsaturatedfattyacylchainsassembleintoahighlyordered,gel-likebilayerthatmaximizeshydrophobicinteractionsbetweentails.• Heat– disordersnonpolartails– inducesatransitionfromageltoafluidwithinatemperaturerangeofonlyafewdegrees(phasetransitiontemperature).• Chaindisorderdecreasesbilayerthickness.

• Mostlipidsandmanyproteinsarelaterallymobile inbiomembranes.• Lipidmoleculemovementinmembrane:• Exchangesplaceswithneighborsinaleaflet~107times/sec.

Howtoshowtheinterchange?

Fluorescencerecoveryafterphotobleaching(FRAP)experimentscanquantifythelateralmovementofproteinsandlipidswithintheplasmamembrane

• Step1:Labelcellswithafluorescentreagentthatbindsuniformlytoaspecificmembranelipidorprotein.

• Step2:Irreversiblybleachwithlaserlightfluorescenceinasmallareaofthecellsurface.• Step3:Measurefluorescencerecoveryinthespotovertime.

• Fluorescenceofthebleachedpatchincreasesasbleachedfluorescentlipids/proteindiffusein(fluorescencerecovery)andbleachedonesdiffuseout.

MajorLipidComponentsofSelectedBiomembranes

Effectoflipidcompositiononbilayerthicknessandcurvature.

• (a)Puresphingomyelin(SM)bilayer:• Thickerthanphosphoglyceride(phosphatidylcholine)bilayer

• Cholesterollipid-orderingeffectincreasesphosphoglyceride bilayerthickness. [Lipidraftsarethickerthanothermembraneregions.]

• (b)Phospholipids:• PCcylindricalshape– formsessentiallyflatmonolayers

• PEconicalshape(smallerheadgroup)–formscurvedmonolayers

• (c)BilayerenrichedwithPCintheexoplasmicleafletandwithPEinthecytosolicface(asinmanyplasmamembranes)– naturalcurvature

• Severalproteinscancurveamembranebybindingtoaphospholipidbilayersurface–importantinformationoftransportvesiclesthatbudfromadonormembrane.

Cholesteroleffect

Sphingomyelin• Sphingomyelin (SPH,ˌsfɪŋɡoˈmaɪəlɪn)isatype

of sphingolipid foundinanimal cellmembranes,especiallyinthemembranous myelinsheath thatsurroundssome nervecell axons.

Effectoflipidcompositiononbilayerthicknessandcurvature.

• (a)Puresphingomyelin(SM)bilayer:• Thickerthanphosphoglyceride(phosphatidylcholine)bilayer

• Cholesterollipid-orderingeffectincreasesphosphoglyceride bilayerthickness. [Lipidraftsarethickerthanothermembraneregions.]

• (b)Phospholipids:• PCcylindricalshape– formsessentiallyflatmonolayers

• PEconicalshape(smallerheadgroup)– formscurvedmonolayers

• (c)BilayerenrichedwithPCintheexoplasmicleafletandwithPEinthecytosolicface(asinmanyplasmamembranes)– naturalcurvature

• Severalproteinscancurveamembranebybindingtoaphospholipidbilayersurface–importantinformationoftransportvesiclesthatbudfromadonormembrane.

Iftoomuchcholesterolinthecellmembrane…

• Whatshouldthecellsdo?

LipiddropletsformbybuddingandscissionfromtheERmembrane.

• Cellsstoreexcesslipidsinlipiddroplets:• Storagecompartmentsfortriglyceridesandcholesterolesters– alsomayserveasplatformsforstorageofproteinstargetedfordegradation.

• Lipiddropletformation:• Cholesterolestersandtriglyceridesaccumulatewithinthehydrophobiccoreofthelipidbilayer.• Delaminationofthetwolipidmonolayersformsa“lens.”• Lensgrowthcreatesasphericaldroplet– releasedbyscissionattheneck.• ThenewlyformeddropletissurroundedbyalipidmonolayerderivedfromthecytosolicleafletoftheERmembrane.

7.2MembraneProteins:StructureandBasicFunctions• Biologicalmembranescontainintegral,lipid-anchored,andperipheralproteins.

• Integralmembraneproteinscontainoneormorehydrophobicmembrane-spanningdomains.

• Transmembraneproteinsandglycolipidsareasymmetricallyorientedinthebilayer.

• Non-ionicdetergentsselectivelyextracttransmembraneproteins.

Integralproteins

• Whatmeanstheintegral?

Thenwhichkindofaminoacids?

Ifithasalpha-helixstructure,whatkindofRgroup?

StructureofglycophorinA,atypicalsingle-passtransmembraneprotein.

• Single(23-residue)membrane-spanningαhelix• composedofaminoacidswithhydrophobic(uncharged)sidechains• αhelixtypically20–25aminoacidslongintransmembraneproteins

Thenthinkaboutthefluid-mosaicmodel

Chargedresiduescanorchestratetheassemblyofmultimericmembraneproteins.

• Tcellreceptor(TCR)forantigen– composedoffourseparatedimers:• αβpairdirectlyresponsibleforantigenrecognition• CD3complexaccessorysubunits– γ,δ,ε,andζsubunits• Electrostaticattractionofpositiveandnegativechargesoneachtransmembranedomainformsthecompletecomplex.

Inthefluid-mosaicmembrane!

Lipidanchoredproteins

• Anchor?

Anchoringofplasma-membraneproteinstothephospholipidbilayerbycovalentlylinkedhydrocarbongroups.

(a)Acylation

• (b)Prenylation:

(c)GPI(glycosylphosphatidylinositol)lipidanchor

Glycolipid:anchoredtothelipidHumanABObloodgroupantigens

• Threestructurallyrelatedoligosaccharidescomponentsofcertainglycoproteinsandglycolipids– onthesurfaceofhumanerythrocytes(RBCs)andothercelltypes

DetergentWhenwillyouusethedetergent?

• Chemicalnature:• Non-ionic– donotdenatureproteins;usefulforsolubilizingintegralmembraneproteins• Ionicdetergents– chargedisruptsionicandhydrogenbondstodenaturemostproteins

• Solubility:• Lowconcentrations– detergentsdissolveinwaterasisolatedmolecules• Higherconcentration– detergentsformmicelles

Structuresoffourcommondetergents.

Whenwillyouusethedetergent?

• Criticalmicelleconcentration(CMC):

7.3Phospholipids,Sphingolipids,andCholesterol:SynthesisandIntracellularMovement• Cellssynthesizenewmembranesbytheexpansionofexistingmembranes.

• Membranelipidscontainsaturated/unsaturatedfattyacidsofvariouschainlengths.

• FattyacidsaresynthesizedinERandmovedtotheotherleafletbyflippases andtoothermembranesbymultiplemechanisms.

• HMG-CoAreductasecatalyzesthecholesterolbiosynthesisrate-controllingstep.

PhospholipidsynthesisintheERmembrane

Membrane-associatedenzymescatalyzethelaststepsofphospholipidsynthesis attheinterfacebetweenamembrane(smoothER) andthecytosol

Cholesterolbiosyntheticpathway

CholesterolissynthesizedbyenzymesinthecytosolandERmembrane(mainlyinlivercells)

Proposedmechanismsoftransportofcholesterolandphospholipidsbetweenmembranes.

Canyouexplainthesemodels???

Discussionwithfriends• Forimmunohistochemistrythemembraneshouldbe

permeabilizedbuttheconformationofproteinshouldbeintact.Whichkindofdetergentisthebestfortheimmunohistochemistryandhowconcentrationisit?

• Findeachexampleproteinthatattachestotheplasmamembranebyprenylation,acylationandGPI.

• FindthegeneralstructureandfunctionofHIVenvelopeglycoprotein.

• Read the ‘aboutcover’andexplainit.

https://www.nature.com/nature/journal/v545/n7653/index.html