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Mutants in Lactose Permease: Effects on Transport and Binding

This slide show presents the effects of mutants in Lactose Permease on transport and binding processes. Mutants E325A and E325D show different characteristics in H+ translocation, substrate binding, and conformational changes. The R302S mutant is specifically defective in lactose/H+ symport. Explore the results and implications of these mutations.

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Mutants in Lactose Permease: Effects on Transport and Binding

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  1. SLIDE SHOW II E325 Mutants

  2. + - - - A. Ground state IX R302 E325 X H322 VIII K319 H+ E269 + D240 R + C 144 VII 148 - V E126 IV

  3. + - - B. Mutant E325A does exchange and counterflow, but no reactions involving H+ translocation IX R302 A325 X H322 VIII K319 H+ E269 + D240 R + C 144 VII 148 - V E126 IV

  4. + - - - C. Mutant E325D is partially uncoupled. IX R302 D325 X H322 VIII K319 H+o E269 + D240 R + C 144 VII 148 - V E126 IV

  5. + - - - - + D. 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

  6. E. Why E325D has lower affinity for ligand

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

  8. G. ... 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

  9. H. 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

  10. + - - - - Si + Si + I. Substrate dissociates to the inside. H+ IX R302 VIII D325 H322 K319 X E269 D240 R C Si 144 VII 148 V E126 IV

  11. + - - - - + + J. Asp325 re-juxtaposes with Arg302, H+ IX R302 VIII D325 H322 K319 X E269 D240 R C 144 VII 148 V E126 IV

  12. K. and the proton is released to the inside with decreased efficiency. + - - - - + + IX R302 D325 X H+i H322 VIII K319 E269 D240 R C 144 VII 148 V E126 IV

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

  14. 100 Efflux Lactose in (%) Exchange 10 0 20 40 60 80 100 120 Time (sec) M. Like Glu325 mutants, R302S [or R302A] permease is specifically defective in lactose/H+ symport, but catalyzes exchange

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