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 37BIOLOGICAL MEMBRANES! Overview biological rolesstructural features!Membrane lipidsgeneral structures aggregation states polymorphism thermal transitions electrical conductivity electrostatic effects molecular dynamics (translational and rotational diffusion, flip-flop) !Membrane proteinscrystallizationoverview of structural featuresstructure/function relations: bacterial photosynthetic reaction centerbacteriorhodopsinBiochemistry 585; Membrane ProteinsReading ListCRYSTALLIZATION*C. Ostermeier & H. Michel, ACrystallization of membrane proteins@, Curr. Opinion Struct. Biol. 7, 697-701 (1997). #C. Ostermeier, S. Iwata, B. Ludwig & H. Michel, AFv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase@, Nature Structural Biology, 2, 842-846 (1995). Optional: *E.M. Landau & J.P. Rosenbusch, ALipidic cubic phases: A novel concept for the crystallization of membrane proteins@, Proc. Natl. Acad. Sci. USA 93, 14532-14535 (1996).[ *pdf files on website; #reprint will be provided ]STRUCTURES AND FUNCTIONSOverview: *S. Scarlata, "Membrane Protein Structure", Chap. 1, Section 2, Biophysical Society on-line textbook. *J.U. Bowie, "Membrane proteins: are we destined to repeat history", Curr. Opinion Struct. Biol. 10, 435-437 (2000). *G.G. Shipley, "Lipids; Bilayers and non-bilayers: structures, forces and protein crystallization", Curr. Opinion Struct. Biol. 10, 471-473 (2000).Electron Transfer Mechanisms:Optional:*J.R. Winkler, "Electron tunneling pathways in proteins", Curr. Opinion in Chem. Biol. 4, 192-198 (2000). #C. C. Page, C.C. Moser X. Chen & P.L. Dutton, "Natural engineering principles of electron tunneling in biological oxidation-reduction", Nature 402, 47-52 (1999).Bacterial Photosynthetic Reaction Center: #U. Ermler, H. Michel & M. Schiffer, "Structure and function of the photosynthetic reaction center from Rhodobacter sphaeroides", J. Bioenerg. Biomembr. 26, 5-15 (1994).*J.P. Allen & J.C. Williams, "Photosynthetic reaction centers", Minireview, FEBS Lett. 438, 5-9 (1998).#N.W. Woodbury & J.P. Allen, APathway, kinetics and thermodynamics of electron transfer in wild type and mutant reaction centers of purple nonsulfur bacteria@, in Anoxygenic Photosynthetic Bacteria, R.E. Blankenship et al., eds, Chap. 24, pp. 527-557, Kluwer Acad. Publ., 1995.Optional: *J. Deisenhofer et al., ACrystallographic refinement at 2.3  resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis@, J. Mol. Biol. 246, 429-457 (1995)].*P.K. Fyfe and M.R. Jones, "Re-emerging structures: continuing crystallography of the bacterial reaction centre", Biochim. Biophys. Acta 1459, 413-421 (2000). *M.Y. Okamura et al., "Proton and electron transfer in bacterial reaction centers", Biochim. Biophys. Acta 1458, 148-163 (2000).]Bacteriorhodopsin: *J.K. Lanyi and H. Luecke “Bacteriorhodopsin”, Curr. Opinion Struct. Biol., 11, 415-419 (2001).#W. Khlbrandt "Bacteriorhodopsin- the movie", Nature 406, 569-570 (2000). Optional: *J.K. Lanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3, Biophysical Society on-line textbook.BIOLOGICAL ROLES OF MEMBRANES SELECTIVE PERMEABILITY BARRIERS (CELL COMPARTMENTALIZATION): PUMPS, GATES SIEVES STRUCTURAL ORGANIZATION OF CELLULAR PROCESSES (ENERGY TRANSDUCTION): RESPIRATION, PHOTOSYNTHESIS, VISION RECEPTORS FOR EXTERNAL STIMULI: HORMONES, NEUROTRANSMITTERS CELL RECOGNITION: IMMUNE RESPONSE, TISSUE FORMATION INTERCELLULAR COMMUNICATION: NERVE IMPULSE TRANSMISSIONMOST MEMBRANES ARE MULTI-FUNCTIONALSTRUCTURAL FEATURES OF MEMBRANES MULTIPLE COMPONENTSLIPIDS (PHOSPHOLIPIDS, GLYCOLIPIDS, CHOLESTEROL):BILAYER STRUCTURE FORMS MAIN PERMEABILITY BARRIER.PROTEINS (PERIPHERAL, INTEGRAL): PROVIDE BOTH STRUCTURAL AND FUNCTIONAL CHARACTERISTICS.CARBOHYDRATE (COVALENTLY BOUND TO LIPID AND PROTEIN): SURFACE RECOGNITION. BROAD COMPOSITIONAL VARIABILITYCORRELATED WITH FUNCTION MOSTLY SELF ASSEMBLINGHYDROPHOBIC AND ELECTROSTATIC FORCES LEAD TO BILAYER FORMATION AND PROTEIN INCORPORATION (CARBOHYDRATE ADDED ENZYMATICALLY AFTER ASSEMBLY) ASYMMETRICINSIDE DIFFERENT FROM OUTSIDE WITH RESPECT TO LIPID AND PROTEIN (CARBOHYDRATE ONLY FOUND ON OUTER SURFACE) DYNAMIC STRUCTUREFLUIDITY, FLEXIBILITY, TWO-DIMENSIONAL DIFFUSIONBIOLOGICAL SIGNIFICANCE OF LIPID POLYMORPHISM POTENTIAL TO FORM NONBILAYER STRUCTURES MAY ALLOW DISCONTINUITIES IN BILAYER AND THEREBY PROMOTE:MEMBRANE FUSION AND VESICLE FORMATION DURING CELL DIVISION.VESICLE-MEDIATED PROTEIN TRAFFICKING.INTEGRATION OF NON-LIPID COMPONENTS INTO MEMBRANE.MOVEMENT OF MACROMOLECULES THROUGH MEMBRANE.LATERAL MOVEMENT OF MACROMOLECULES.STABILIZATION OF MEMBRANE PROTEIN COMPLEXES.CONFORMATIONAL INTERCONVERSIONS ASSOCIATED WITH PROTEIN FUNCTION.TRANSLATIONAL DIFFUSION IN MEMBRANES USUALLY MEASURED BY FRAP (FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING) USING FLUOROPHORE-LABELLED LIPIDS. INVOLVES PHOTOBLEACHING A SMALL REGION OF MEMBRANE SURFACE WITH LASER AND MEASURING TIME DEPENDENCE OF MOLECULAR DIFFUSION INTO BLEACHED AREA. Dtrans (translational diffusion coefficient) RELATED TO MEAN SQUARE DISPLACEMENT: _r2  4 Dtrans t FOR BOTH LIPIDS AND PROTEINS, Dtrans  10-8 cm2s-1 at 25 °C. THUS, IN 1 SECOND: _ r2 = 4 x 10-8 cm2 _(r2)1/2 (MEAN DISPLACEMENT) = 2 x 10-4 cm = 2 microns (i.e. MOVEMENT IS RAPID).MEASUREMENT OF MEMBRANE FLUIDITY AND MOLECULAR ROTATION BY FLUORESCENCE DEPOLARIZATIONUSE A COVALENTLY ATTACHED FLUOROPHORE, OR A FLUORESCENT PROBE WHICH PARTITIONS INTO THE BILAYER (e.g. DPH; DIPHENYLHEXATRIENE). EXCITE WITH POLARIZED LIGHT AND MEASURE POLARIZATION OF FLUORESCENCE. IF FLUOROPHORE ROTATES DURING EXCITED STATE LIFETIME, FLUORESCENCE WILL BECOME DEPOLARIZED.DEFINITIONS:P = POLARIZATION = (I - I) / (I + I) r = ANISOTROPY = (I - I) / (I + 2I) PERRIN EQUATION:r0