Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Nutrient Transport in E. coliPassive transporters : Most nutrients < 600 Da - PorinsLigand Gated Porins - sugarsActive transporters : Larger nutrients such as iron and cobalamin (vitamin B12 : CNCbl) BtuB, FecA, FhuA, and FepA etc. FhuAFhuDFhuB/CBtuFBtuC/DFerrichrome Vitamin B12BtuBProposed TonB Dependent Transport CycleInnerMembraneH+Cytoplasmic SpaceCNCblBtuBCNCblA BCNCblCNCblTon boxTonBExbBExbDC D APeriplasmicSpaceExtracellular SpaceOuterMembraneEPR Spectra for 2 consecutive strands of the barrelQ D T S P T L VD V A N R F E Q P R S T V L A P TT VV T R QLIAGSTMHKRNQVDD I D R W QTSVNDVLRRLSPGVDITQNGGSGQLSSIRGTNASHVLVLIDG V R L N L A G V S G S A D L S Q P I A L V Q RRTTIIN V VV E Y I R G P R S A VGSDAIG GDGPSITAESNSDVNEGGWQQQQTSDRLAGKGPADVNEGSLGKTVTDHTHLTLRLHDASTFGGNRTNETSKADGDLHGNRNILKQLISSSDQQTRLGKPMVNEGVTD TRLGGRTNESGKAGDHGNRNSDHEDLTSASDRGVIVGHGIAQKQIQGTQQQTLVTTGLFAGSIVESRAGKAEGTGNRNSAFQKKISKIAKNQDTTDDQQQVAGAGKTQAKLIPDLTLSAGTVAERS VGKYTAILHSTVLDGVEA LGE IEGGSLFGDDNTQLSTKNDVVAVGPTTLPGDTQRVAAAGSDQSLVANEQPRSTLAPTNGGGLSIRGTNGVSGSADLPAEDDPQDTPTDREPGQLRGTVLDGRLNLGVGSADLSDDDDTDDTDKDTT1245 36791081113141516171819202122L11L10L9L8L7L6L5L4L3L2L112F YWWWWWWWWWWWFFFFFFFFYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY YYYYYYYYYYYYFYH135162187214240264285325343304361381403424444465490512534557576F594TYYFanucci et al. Biochemistry, 41, 11543, 2002•Pulsed EPR - direct T1 measurementsVs•Continuous Wave Power Saturation - indirect T1 measurementsEnhancements in relaxation due to paramagnet tell you about collisional frequency and hence accessibilityStrategy: Add a paramagnetic species that can collide with spin label~ relative enhancements in relaxation timesAccessibility is measured via Relaxation MeasurementsPower Saturation of the EPR Resonance AmetaloxygenmembraneA = I•P1/2 •[1 + (21/ - 1)P/P1/2]-oxy = P´1/2oxy - P´1/2nitmet = P´1/2met - P´1/2nitaccessibility 0 2 4 6 8 10 Sqrt (P)nitoxymetP1/2 = 5.6P1/2 = 10.6P1/2 = 19.1-2024-40 -20 0 20 40Distance from phosphate (A)AqueousHydrocarbonNitroxide Accessibility and Membrane Depth from EPR = ln (P1/2oxy/P1/2metal)Calibration Points: bR and DOXYL-PCPDB ID: 1ap9 5-DOXYL +1.5 8.1 7-DOXYL +2.1 10.510-DOXYL +2.4 14.012-DOXYL +2.8 16.0 bR109R1 +2.2 11.0 bR116R1 +4.1 20.5 bR117R1 +4.2 22.5 bR124R1 +2.0 10.5Sample DistanceFrazier, A.A. et al. Biochem, 42 (2003), 96-105-2024-40 -20 0 20 40Distance from Phosphate (A) A B C Dt a n h d i s t a n c e -C2 domaindoxyl-PCbacteriorhodopsinModeling of C2A on the Membrane Surface174175173176142202171201240170229239231236233197197b199237234Frazier, A.A. et al. Biochem, 42 (2003), 96-105OOOOHOHNOONOOOOONiH2OOH2BtuB as a Ruler From the Bilayer SurfaceDOGS-NTA-NiNi(II)Ni(II)EDDAca. 14 Å- 2 0 - 1 0 0 1 0 2 0 3 0- 20246 a q u e o u sD is ta n c e f r o m P h o s p h a te s ( Å )m e m b r a n e1 2840P1/2 DOGS-Ni-NTAO2N i ( I I ) E D D Aa q u e o u sO2O2D O G S - N i - N T Am e m b r a n eBtuB as a Ruler From the Bilayer SurfaceNi(II)Ni(II)EDDAca. 14 Å = ln (P1/2oxy/P1/2metal)ConclusionsKcsANi(II)EDDADOGS-Ni-NTABtuBbRC2A CPLA2~ 14ÅO2SDSL “Rulers”GM2AP oligomerizationGM2APA60~ 2ÅPOPC + BMPLipid Composition Affects BindingSecondary Structure Analysis: Spin Label MappingTransmembrane Water Filled Pores-helix bundlemembrane-barrelmembraneaqueous phasemembraneaquaccessibilityoxygenmetal complexoxygenmetal complex 2 4 6 8 2 4 6 8Power Saturation of EPR Spectra for 2 consecutive putative strands of the -barrel -0.2-0.10.00.10.2178 176 174 172 170 168 166 164 1620.00.40.81.21.62.0 -0.2-0.10.00.10.2146 148 150 152 154 156 158 160 162 1640.00.40.81.21.62.0Ni(acac)2 accessibilityOxygen accessibilityresidue-barrelmembraneaqueous phasemembraneaqu26 of 36Power Saturation of EPR Spectra for 2 consecutive putative strands of the -barrel -0.2-0.10.00.10.2178 176 174 172 170 168 166 164 1620.00.40.81.21.62.0 -0.2-0.10.00.10.2146 148 150 152 154 156 158 160 162 1640.00.40.81.21.62.0Ni(acac)2 accessibilityOxygen accessibilityresidue172 170 168 166 164 162-2-10123-2-10123148 150 152 154 156 158 160 ResidueResidueaqueousmembraneFanucci et al. Biochemistry, 41, 11543, 200227 of 36Bottom ViewSide ViewExtracellularPeriplasmicLipid BilayerTonbox(6-12)S1 (25-31)extendedtonbox (13-17)Structure of BtuB• 66 kD protein• beta barrel of 22 antiparallel strands• globular hatch/core domainChimento et al. NSB 2003 (10) 394-4017 of
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