2. SLIDE SHOW I Wild-type mechanism

3. K 319 - - + - + C E 325 H 322 D 240 R E 126 148 144 IV VII V - E 269 VIII + R 302 IX X A. In the ground state the permease is protonated H+H+

4. K 319 - + - + C E 325 H 322 D 240 R 148 144 - E 126 IV VII V - E 269 VIII + R 302 IX X B. Substrate binds from the outside H+oH+o SoSo

5. K 319 - - + - + C E 325 H 322 D 240 R E 126 148 144 IV VII V - E 269 VIII + R 302 IX X SoSo C. Binding of substrate causes a conformational change disrupting the interaction of Glu269 and His322 and …. H+H+

6. + - + C R R 302 E 126 148 144 IV VII V IX - D 240 - E 325 H 322 K 319 + X - E 269 VIII SiSi D.... causes substrate to become accessible to the internal surface, and the proton moves to His322/Glu325. H+H+

7. + - + C R R 302 E 126 148 144 IV VII V IX - D 240 - E 325 H 322 K 319 + X - E 269 VIII SiSi E. Rotation of Helix X continues towards the hydrophobic phase of the membrane, H+H+

8. + - + C R R 302 E 126 148 144 IV VII V IX - D 240 - E 325 H 322 K 319 + X - E 269 VIII SiSi F. raising the pK a of Glu325. H+H+

9. + + C R R 302 148 144 IV V IX - - E 126 VII D 240 - E 325 H 322 K 319 + X - E 269 VIII G. Substrate dissociates to the inside, H+H+ SiSi SiSi

10. H. And Glu325 re-juxtaposes with Arg302. + - + C R R 302 E 126 148 144 IV VII V IX - D 240 - E 325 H 322 K 319 + X - E 269 VIII H+H+

11. K 319 - + - + C E 325 H 322 D 240 R 148 144 - E 126 IV VII V - E 269 VIII + R 302 IX X I. The proton is released to the inside, H+iH+i

12. K 319 - - + - + C E 325 H 322 D 240 R E 126 148 144 IV VII V - E 269 VIII + R 302 IX X J. the permease relaxes into the ground state and becomes protonated from the outside. H+oH+o

13. K. Crosslinking between Asp240 (helix VII) and Lys319 (helix X): Effect of ligand binding MTS-1-MTS: 3 Å MTS-3-MTS: 5 Å; flexible o-PDM: 6 Å; rigid p-PDM: 10 Å; rigid