1Membrane lipidsCholesterolPhospholipidsSphingolipidsBilayers, micelles and liposomes2Packing of LipidsDetergentsSDSDOCNP40!OGCHAPS3X-ray Diffraction of Lipid Bilayers &Biological MembranesDifferentialScanningCalorimetryElectronSpinResonance4Lipid Rafts - one example of membrane mosaicismPatches of less fluid membrane enriched insphingolipids & cholesterolDetergent-insolubleLess dense than bulk membraneTEM - osmium tetroxide stainFreeze FractureTEM - metal shadowing~ 2 nm~ 2 nmIMPs - intramembranousparticles - integral membraneproteins5Peripheral membraneproteins - easily removedwithout detergentIntegral membraneproteins - only dissolvedand handled in detergentPeripheral and IntegralMembrane ProteinsReconstitutionProtein Folding in MembranesInside of membrane is hydrophobicResidues contacting lipid side chains need to be hydrophobicThere is an energy cost associated with introducing chargedor hydrophilic residues into the hydrophobic bilayer.On balance "G must be < 0Free energy balance sheet - some examples20 x F residues ~ 30 kcal/mole GAINIonized group ~ 25-40 kcal/moleIon pair ~ 10-15 kcal/moleHydroxyl group ~ 4 kcal/moleOne unsatisfied H bond ~ 5 kcal/moleTherefore - a 3-4 residue turn ~ 15-20 kcal/mole6Protein Folding in Membranes#-helix 3.6 residues per turn, 1.5 A/residue20-22 residues to cross membraneH-bonds satisfied within one helixOften a hydrophobic stretch!-sheet 2 residues per turn, 3.3 A/residue7-9 residues to cross membraneH-bonds only satisfied in multiple strandsRarely detectable as a hydrophobic stretch#-helix!-sheetTransmembrane segments7Side viewsPlan viewsHydropathy plotsMembrane Proteins8BACTERIAL RHODOPSINX-ray structureEM structureSide viewsPlanviewAQUAPORINMurata et al Nature 2000monomer - side viewtetramer - top viewtetramer - side view9Cyanobacterial Photosystem IBacterial porins10Nicotinic Acetylcholine ReceptorStructure determined by electronCrystallography to 4A resolutionMiyazawa, Fujiyoshi & UnwinNature 423: 949-955 (2003)Unwin, J.Mol.Biol. 346:967-989 (2005)Each subunit contributes an #-helixto the pore and three to the surroundExtracellular domain is homologouswith an acetylcholine-binding protein from a snailNicotinic Acetylcholine ReceptorStructure determined by electronCrystallography to 4A resolutionMiyazawa, Fujiyoshi & UnwinNature 423: 949-955 (2003)Unwin, J.Mol.Biol. 346:967-989 (2005)Binding of AChcauses 15o rotation and opens gate inmiddle of membrane11#-helix!-sheetMaybe thetranslocon makes thisdetermination?Predicting TransmembraneTopography“Positive-inside” ruleNilsson and von HeijneCell 62:1135-1141 (1990) OUTINOUTIN12Testing TransmembraneTopographyOUT INProteolysisAntibodiesSurface LabelingSurface Labeling ReagentsNH2-PROTEINBIOTIN(sulfo)NHS-BIOTINN-O-C-(CH2)4-OOSNHNH=O=OI*+N=N-__-SO3Diazotized Iodo-sulfanilic acidSCN-_-SO3-C=C-_-SO3_-NCSDiisothiocyanostilbene-2,2’ disulfonate(DIDS)UVI*CCF3..activation toreactive carbeneI*CN=NCF3Trifluoro methyl-3 iodoaryldiazirine(125I-TID)hydrophobic, membrane solubleunreactive-SO313Testing TransmembraneTopographyLehmann et alJBC 272:12047-51 (1997)Evidence for a six transmembranedomain structure of presenilin 1OUT INLipid Modifications of Membrane Proteins #1A. Phosphatidyl inositol glycan (PIG-tailed, PI-linked)• Attached near C-terminus - signalled by a short hydrophobic stretch which is removed.• Linkage is via ethanolamine to carboxyl terminus-amide bond; irreversible• Anchors external membrane proteins in external leaflet via two acyl chains.• Connection can be cleaved by PI-specific phospholipase C.• This is only known external connection - others provide insertion into internal leaflet.B. Myristoylation• 14C fatty acid (myristate) attached via amide bond to N-terminal glycine.• There is a recognition sequence internal to N-terminal glycine.• Amide linkage is irreversible.• Confers some affinity for membranes (~8kcal/mole, equivalent to inserting 10 CH2 in the hydrophobic base Kd $ 10-4 M).• This is insufficient for efficient membrane localization. • Myristoylation is usually coupled with other elements conferring membrane-bindingeg, basic amino acids or palmitoylation.14Lipid Modifications of Membrane Proteins #2C. Palmitoylation• Attachment of 16C fatty acid (palmitate) to cysteine residue via S-acyl linkage - reversible.• Often associated with irreversible modification (myristoylation or prenylation) at nearby residues.• Palmitoylation adds membrane-binding affinity.D. Prenylation• Attachment of a prenyl group(isoprenoid) to a cysteine residue very near the C-terminus. • The C is located in the sequence CAAX (or variants thereof) where X is any amino acid and A is a hydrophobic residue. The AXX sequence is removed by proteolysis and the carboxyl of the cysteine is methylated.• The linkage is via a thioether (-CH-S-) bond and is irreversible.• Two prenyl groups are found: farnesyl (15C) or, more commonly, geranyl geranyl (20C)attached by different prenyl transferases recognizing different CAAX sequences.• Prenylation is frequently associated with palmitoylation at nearby cysteines.farnesyl (15C)geranyl geranyl (20C)Multiple ways for membrane proteins to associate with the membraneLipid-linkedPeripheral - binding to phospholipid head groupsPeripheral - binding to integral proteinsIntegral- partially inserted- single pass TM- multiple pass TM15The not-so-fluid mosaic membraneEngelman DM (2005) Membranes are more mosaic than fluid. Nature 438:578-580. Many proteinoligomersMany membraneproteins occlude lipidBilayer thickness variesLateral Mobility of Membrane Proteins16Fluorescence Recovery afterPhotobleaching (FRAP)17Restrictions on Lateral Mobility ofMembrane ProteinsSingle particle tracking andoptical tweezers revealrestrictions on lateralmobility of integralmembrane proteins.Submembrane cytoskeletonof red blood cellsProtein CHO18Dissection of the Erythrocyte MembraneDissection of the Erythrocyte MembraneDetergent extracts - Band 3, glycophorin - INTEGRALLow salt extracts - Spectrin (1,2) and actin (5)High salt extracts - ankyrin (2.1, 2.2) and band 4.1- PERIPHERALGHOSTSSKELETONS19SpectrinHuman mutations in spectrin, ankyrin and band 3 produce defective red cells2-ply Organization ofErythrocyte MembranesLux and Palek 1995How relevant is this to other membranes?20Restrictions on Lateral Mobility ofMembrane Proteins in Other CellsEM analysis of submembranous cytoskeletonMorone…Kusumi et al J. Cell Biol. 2006EM tomography of freeze,