1. SLIDE SHOW III
Rescuing E325D

2. A. Mutant E325D is partially uncoupled and has lower KD
for substrate. IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148
VII - V
E126 IV

3. B. Substrate binds from the outside with lower affinity.IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148 So
VII - V
E126 IV

4. H+o IX R302 + - D325 X H322 VIII K319 - E269 + - D240 R + C 144 148
C. Binding of substrate causes a conformational change disrupting the interaction of Glu269 and His322, …. IX
R302 + -
D325 X
VIII
H322
H+o
K319 -
E269 + -
D240 R + C
144
148 So
VII - V
E126 IV

5. H+ IX R302 + - VIII D325 H322 X K319 E269 + - - D240 R + C 144 148 VII
D. ... causes substrate to become accessible to the internal surface, and the proton moves to His322/Asp325. IX
R302 + - H+
VIII
D325
H322 X
K319
E269 - + -
D240 R + C
144
148 Si
VII - V
E126 IV

6. H+ IX R302 + - VIII D325 H322 X K319 E269 + - - D240 R + C 144 148 VII
E. Rotation of Helix X continues towards the hydrophobic phase of the membrane, IX
R302 + - H+
VIII
D325
H322 X
K319
E269 - + -
D240 R + C
144
148 Si
VII - V
E126 IV

7. H+ IX R302 - + VIII D325 H322 K319 E269 X + - - D240 R + C 144 148 VII
F. raising the pKa of Asp325. IX H+
R302 - +
VIII
D325
H322
K319
E269 X + - -
D240 R + C
144
148 Si
VII - V
E126 IV

8. G. Substrate dissociates.IX H+
R302 - +
VIII
D325
H322
K319
E269 X + - - Si
D240 R + C
144 Si Si
148
VII - V
E126 IV

9. H+ IX R302 - + D325 VIII H322 K319 X E269 + V316G/A - - D240 R + C 144
H. By introducing a Gly or Ala residue at the periplasmic end of helix X, Asp325 re-juxtaposes with Arg302 without improving affinity for substrate. H+ IX
R302 - +
D325
VIII
H322
K319 X
E269 +
V316G/A - -
D240 R + C
144
148
VII - V
E126 IV

10. I. The proton is released to the inside with increased efficiency, …..IX
R302 + -
D325 X
H322
H+i
VIII
K319 -
V316G/A
E269 + -
D240 R + C
144
148
VII - V
E126 IV

11. H+o IX R302 + - D325 X H322 VIII K319 - V316G/A E269 + - D240 R + C
J. and the permease relaxes into the ground state and becomes protonated from the outside. IX
R302 + -
D325 X
H322
VIII
K319
H+o -
V316G/A
E269 + -
D240 R + C
144
148
VII - V
E126 IV