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This slideshow presentation delves into the intricate details of the rescued mutant E325D, exploring its uncoupled nature and altered substrate affinity. Witness the conformational changes leading to proton movement and enhanced efficiency in this elucidating journey.
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SLIDE SHOW III Rescuing E325D
A. Mutant E325D is partially uncoupled and has lower KD for substrate. + - - - IX R302 D325 X H322 VIII K319 H+o E269 + D240 R + C 144 VII 148 - V E126 IV
+ - - - - + + B. Substrate binds from the outside with lower affinity. IX R302 D325 X H322 VIII K319 H+o E269 D240 R C 144 VII So 148 V E126 IV
C. Binding of substrate causes a conformational change disrupting the interaction of Glu269 and His322, …. + - - - - + IX R302 D325 X H322 VIII K319 H+o E269 + D240 R C 144 VII So 148 V E126 IV
D. ... causes substrate to become accessible to the internal surface, and the proton moves to His322/Asp325. + - - - IX R302 H+ D325 VIII H322 X K319 E269 + D240 R + C 144 Si VII 148 - V E126 IV
E. Rotation of Helix X continues towards the hydrophobic phase of the membrane, + - - - IX R302 H+ D325 VIII H322 X K319 E269 + D240 R + C 144 Si VII 148 - V E126 IV
+ - - - F. raising the pKa of Asp325. H+ IX R302 VIII D325 H322 K319 X E269 + D240 R + C 144 Si VII 148 - V E126 IV
- - - G. Substrate dissociates. H+ IX R302 + VIII D325 H322 K319 X E269 + - Si D240 R + C Si Si 144 VII 148 V E126 IV
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 VII 148 - V E126 IV
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 VII 148 V E126 IV
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 VII 148 - V E126 IV