lecture 19 - 19-1MCB 354Lecture 19Solubilization of...

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Unformatted text preview: 19-1MCB 354Lecture 19Solubilization of Membrane ProteinsMembrane DomainsMembrane TransportReading in Lehninger:Chapter 11, pp. 384-386; 389-391; 391-396Solubilization of integral membrane proteins:principle: 19-2Solubilization 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-POLAR19-3Dissociation of membranes in detergent: stagesow Detergent:membrane P-Lipid ratio (0.1-1:1)detergent monomers incorporate into bilayer,membrane more fluid & permeable,but not disrupteds D:Lratio increases,membrane becomes saturated with detergent,detergent micelles begin to formLipid molecules begin to be incorporated intomicelles (at D:Lratio of ~ 2:1)More and more lipid solubilized(incorporated into mixed D-Lmicelles)Solubilization complete (D:L~ 3:1)igh D:L(> 4:1)More detergent incorporated into mixed micellesD:Lmicelles become smallerMainly peripheral proteins extracted,asProtein-Detergentcomplexes( Lipid) Most proteins solubilized asmembrane starts dissociating.Proteins obtained primarily asLipid-Protein-DetergentcomplexesProteins obtained asProtein-Detergentmicelles.Usually 1 Protein/Micelle(unless two or more membrane proteinsinteract to form a complex)What happens to the membrane? What happens to the proteins? 19-4Membrane proteinsHard to crystallize..Have both hydrophilicand hydrophobicregionsNot soluble in aqueous buffer solutions,but traditional crystallization techniques use aqueous solventsSometimes, certain detergents can be used to solubilize a membraneprotein in its native conformation:.....get crystals of protein-detergent complex(very hard to find the right conditions)19-5Fluid mosaic model for membrane organization Model suggests random distribution of membrane lipids and proteins, but some types oflipid may self-associate into domains with potential to cluster certain proteins as well....
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lecture 19 - 19-1MCB 354Lecture 19Solubilization of...

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