4. Nutrient Transport in E. coli Passive transporters : Most nutrients < 600 Da - Porins Ligand Gated Porins - sugars Active transporters : Larger nutrients such as iron and cobalamin (vitamin B 12 : CNCbl) BtuB, FecA, FhuA, and FepA etc. FhuA FhuD FhuB/C BtuF BtuC/D FerrichromeVitamin B12 BtuB

5. Proposed TonB Dependent Transport Cycle Inner Membrane  H + Cytoplasmic Space CNCbl BtuB CNCbl AB Ton box TonB ExbB ExbD CDA Periplasmic Space Extracellular Space Outer Membrane

6. EPR Spectra for 2 consecutive strands of the barrel Fanucci et al. Biochemistry, 41, 11543, 2002

7. Pulsed EPR - direct T1 measurements Vs Continuous Wave Power Saturation - indirect T1 measurements Enhancements in relaxation due to paramagnet tell you about collisional frequency and hence accessibility Strategy: Add a paramagnetic species that can collide with spin label ~ relative enhancements in relaxation times Accessibility is measured via Relaxation Measurements

8. Power Saturation of the EPR Resonance A metal oxygen membrane A = IP 1/2 [1 + (2 1/  - 1)P/P 1/2 ] -   oxy = P´ 1/2 oxy - P´ 1/2 nit  met = P´ 1/2 met - P´ 1/2 nit accessibility Sqrt (P) nit oxy met P 1/2 = 5.6 P 1/2 = 10.6 P 1/2 = 19.1 Nitroxide Accessibility and Membrane Depth from EPR  = ln (  P 1/2 oxy/  P 1/2 metal)

9. Calibration Points: bR and DOXYL-PC PDB ID: 1ap9 5-DOXYL+1.5 8.1 7-DOXYL+2.110.5 10-DOXYL+2.414.0 12-DOXYL+2.816.0 bR109R1+2.211.0 bR116R1+4.120.5 bR117R1+4.222.5 bR124R1+2.010.5 Sample  Distance Frazier, A.A. et al. Biochem, 42 (2003), 96-105

10. C2 domain doxyl-PC bacteriorhodopsin Modeling of C2A on the Membrane Surface 174 175 173 176 142 202 171 201 24 0 170 229 239 231 236 233 197 197b 199 237 234 Frazier, A.A. et al. Biochem, 42 (2003), 96-105

11. BtuB as a Ruler From the Bilayer Surface DOGS-NTA-Ni Ni(II) Ni(II)EDDA ca. 14 Å

12. BtuB as a Ruler From the Bilayer Surface Ni(II) Ni(II)EDDA ca. 14 Å  = ln (  P 1/2 oxy/  P 1/2 metal)

13. Conclusions KcsA Ni(II)EDDA DOGS-Ni-NTA BtuB bR C2A CPLA2 ~ 14Å O2O2 SDSL “Rulers” GM2AP oligomerization GM2AP A60 ~ 2Å POPC + BMP Lipid Composition Affects Binding

14. Secondary Structure Analysis: Spin Label Mapping Transmembrane Water Filled Pores  -helix bundle membrane  -barrel membrane aqueous phase membrane aqu accessibility oxygen metal complex  oxygen metal complex  2468 2468

15. Power Saturation of EPR Spectra for 2 consecutive putative strands of the  -barrel  -barrel membrane aqueous phase membrane aqu 26 of 36

16. Power Saturation of EPR Spectra for 2 consecutive putative strands of the  -barrel Residue   aqueous membrane Fanucci et al. Biochemistry, 41, 11543, 2002 27 of 36

17. Bottom ViewSide View Extracellular Periplasmic Lipid Bilayer Tonbox (6-12) S1 (25-31) extended tonbox (13-17) Structure of BtuB 66 kD protein beta barrel of 22 antiparallel strands globular hatch/core domain Chimento et al. NSB 2003 (10) 394-401 7 of 20