All cells have membranes eukaryotic cells have intracellular membranes • Organelles with 2 • Organelles with a Molecular Membranes or more single membrane Biology membranes • endoplasmic • nucleus (2) reticulum • mitochondria (2) • golgi complex • chloroplasts (3) • endosomes • peroxisomes • lysosomes Types of Membrane Proteins • Integral membrane proteins • Lipid-linked proteins • Peripheral membrane proteins Integral Membrane Proteins • 20 - 30% of total proteins • 60% of drug targets • Essential functions – Transfer information – Transport material – Energy conversion • Few Structures available (~100 helical membrane proteins) Alberts, Molecular Biology of the Cell (1994) p.486. Integral Membrane Protein difficulties: Membrane protein topology • Low abundance • Number of transmembrane segments • Instability outside their native membranes • Their orientation relative to the membrane • Inefficient refolding • Ability to grow ordered 2- or 3-D crystals Advances have largely been made with bacterial proteins Hydrophobicity Scale Membrane protein Explorer ‘Mpex’ http://blanco.biomol.uci.edu/mpex/ Branden & Tooze, p. 245. MacKinnon (2005) Science, pp. 1425 - 1426 Hydrophobicity plots for chains L & M of Reaction center Rhodocater sphaeroides 19 aa window Kyte & Doolittle Hydrophobicity scale Branden & Tooze p.246 Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Fig. 1. Topology mapping using the HA/Suc2/His4C reporter Kim, Hyun et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11142-11147 TIBS (2000) 25:433 Copyright ©2006 by the National Academy of Sciences Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Test of Membrane topology Bacteriorhodopsin Bovine Ca2+-ATPase • Antibody Epitopes • Proteolysis • Modification – Chemical E. coli ClC Cl-/H+ antiporter6 – Glycosylation Von Heijne, Nature Reviews Molecular Cell Biology 7, 909-918 (2006) Hessa, T. Nature 2005 Jan 27;433(7024):377-81. TYPE A SURFACTANTS Lodish, Molecular Cell Biology (2000) p.84 Helenius & Simons (1975) BBA 415:29-79 Type A surfactants (continued) Helenius & Simons (1975) BBA 415:29-79 Helenius & Simons (1975) BBA 415:29-79 GEL FILTRATION Lipid composition of membrane preps (%) -1 Rs = ao + bo (erf )(1-Kd) Rs = Stokes’ radius = constants of gel matrix used ao, bo erf-1 = inverse error function = partial coefficient= (V -V )/V -V ) Kd e 0 t 0 where Ve, V0 and Vt are elution times protein, void and total volume of column, respectively. SUCROSE GRADIENT CENTRIFUGATION AND MOLECULAR WEIGHT . M* = s20,w RT/D20,w (1-v* ps) = sedimentation coefficient, normalized to 20˚C and water Rate-zonal s20,w Centrifugation D = diffusion constant of complex, normalized to 20˚C and 20,w water, can be measured in an analytical ultracentrifuge or it may be replaced by the following expression with Stokes’ radius Rs as a parameter: D= kT/6!nRs where n viscocity of the solvent v* = partial specific volume of complex ps = density of the solvent = gas constant, absolute temperature Lodish, Molecular Cell Biology (2000) p.86 R,T Detergent solubilization of integral membrane proteins Photosynthesis rxn center Purple bacterium Caffrey (2000) Cur. Op. Struct. Biol. 10:486 Branden & Tooze p.237 MacKinnon (2003) Febs Letters KcsA potassium channel from the bacterium Streptomyces lividans 555: 62-65 MacKinnon (2003) Febs Letters 555: 62-65 Integral Membrane Protein Folding Bacteriorhodopsin 1st membrane protein to be completely denatured and refolded in vitro (London & Khorana JBC (1982) 257:7003). Synthetic peptides 3 21-residue peptides that form transmembrane alpha-helices can aggregate/assemble and conduct ions (Lear, Wasserman & DeGrado Science (1989) 240:1437 KcsA MthK (Ca++) MacKinnon (2003) Febs Letters 555: 62-65 Bacteriorhodopsin Folding Models Engelman et al., (2003) FEBS letters 555:122-125 Curran 1999 Transmembrane Regions Integral membrane proteins "-Helices • Type I Single transmembrane span, N- terminus in the ectodomain, C- terminus in the cytosol • Type II Single span, C-terminus in the ectodomain, N-terminus in the cytosol #-barrels • Type III Multiple spans porin from the outer membrane of Rhodobacter capsulatus.