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UIUC MCB 450 - 450 Sp15 Lect 9 for posting-1

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PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48MCB 450Lecture 9Membrane Proteinsperipheralintegrallipid-anchoredMembrane Transport9-1Fluid mosaic model for membrane organization 9-2Membranes are full of proteinsDemonstration/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 + mercaptoethanoland separation by SDS-PAGE)• 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 ways9-3Association of proteins with membranes-1 Membrane proteins are distinguished operationally by the conditions requiredto release them1) Integral: removable only by agents that interfere with hydrophobic interactions (i.e. detergents)= proteins that pass throughmembane at least onceMost “membrane proteins”fall into this category:~1/3 of the different proteinsin most cells!9-4INSIDE CELLAssociation of proteins with membranes-2 9-5OUTSIDECELL2. Peripheral: releasedby relatively mild treatmentsthat interfere w. electrostaticinteractions 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 releasedby treatment with phospholipase C: cleaves a “glycosylphosphatidylinositol” (GPI)membrane anchor attached at COOHterminus of some proteins(other types of protein “lipidation” too)Integral membrane proteins-1 9-61. 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” possible2. A given “transmembrane protein” always has the same orientation in the membraneExample: glycophorin (a protein from the red cell membrane)i) extracellular domain with covalently attached sugarsii) stretch of hydrophobic  buried in core of membraneiii) C-terminal domain in cytoplasmExample: -adrenergic receptor (in plasma membrane)i) N-terminal extracellular domain with covalently attached sugarsii) 7 stretches of hydrophobic  buried in core of membraneiii) hydrophilic loops of varying length between hydrophobic segmentsiv) C-terminal domain in cytoplasm Integral membrane proteins-29-7Transmembrane domains: properties-1 9-81. Transmembrane domains are usually -helical segments of  with non-polar side chains.• Segments long enough to span the bilayer2. Why an -helix? • Internal H-bonding between polar CO-NH groups of peptide bondsin -helix lowers energetic cost of transferring theminto 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 lipidsand there are no H2O molecules w. which to form H-bonds.Transmembrane domains: properties-2 9-93. Length of a helix = 1.5 Å per ,width of hydrophobic core of bilayer ~ 30 Å, so a helix of 20 - 25  would span the thickness of the bilayer.X-ray structure of bacteriorhodopsin:7 transmembrane -helices @ ~20hydrophobic , clustered together:space around and betweenfilled by membrane lipidsA particular class of  may be enrichedon one side of an -helix9-10In 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 side100°100°100°NON-POLARRemember from Lect. 3:Each  is related to nextby a rise of 1.5 Åand a rotation of 100° so, 3.6 per 360° turn,rising at 1.5 Å per Amphipathic helices often form bundles:often seen in transport proteins9-11- 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)Transmembrane segments can be predicted:"hydropathy" plots9-121. Give each  side chain a score according to its relative hydrophobicity- Phe gets high, +ve, score, Arg a low, -ve, score2. 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).Hydropathy plots identify -helicaltransmembrane segments9-134. A stretch of ≥ 20  with a high hydropathy index presumed to bean -helical transmembrane segmenta.k.a. membrane spanning domainTypically, L, I, V, M, F, A, (W, Y at membrane-aqueous interface)Kent and Robertson BMC Evolutionary Biology 2009 9:41 HYDROPHOBICHYDROPHILICExamples of hydropathy plots1 TM domain 7 TM domainsABOVE ~1.5 INTHIS ANALYSIS 9-14COVALENTLY ATTACHEDCARBOHYDRATES NOTINCLUDED IN THEANALYSIS-barrel membrane proteins9-15Non-polar side chains facemembrane interior-sheets curl up to formhollow cylinder, whichcan serve as a porehttp://sbcb.bioch.ox.ac.uk/bond.php-barrel membrane proteins are usually not identifiable usinghydropathy analyses for membrane-spanning -helices(not easy to identify from sequence)9-16Hydropathy plot9-17REPLACE LIPIDSW. DETERGENTSSolubilization of integral membrane proteins:principle:Solubilization of integral membrane proteins:different detergents can be used 1. Strong ionic detergents such as SDSunfold the entire protein by binding to internal core:SDS often denatures proteins2. Non-ionic detergents such as Triton X-100bind mainly to the membrane-spanningsegments of proteins: can allow active protein to be recovered.[polyoxyethylene octyl phenyl ether]MORE POLARNON-POLARPOLARNON-POLAR9-18It's harder to get 3-D structures of membrane proteinsHard to crystallize…..• Have both hydrophilic and hydrophobic regions• Not soluble in aqueous buffer solutions,but


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UIUC MCB 450 - 450 Sp15 Lect 9 for posting-1

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