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Bacteriorhodopsin mutants D85N, D85T and D85,96N as proton pumps
J Tittor1, D Oesterhelt, E Bamberg
1Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
Biophysical Chemistry
|September 1, 1995
Summary
Bacteriorhodopsin mutants D85N, D85T, and D85,96N exhibit altered proton translocation. Azide enhances proton pump currents in D85N and D85,96N, suggesting a modified proton pathway.
Area of Science:
- Biophysics
- Membrane Protein Function
- Photochemistry
Background:
- Bacteriorhodopsin (BR) is a light-driven proton pump.
- Mutations can alter BR's proton translocation mechanism.
- Understanding these alterations is key to elucidating proton transport pathways.
Purpose of the Study:
- To investigate proton translocation in BR mutants D85N, D85T, and D85,96N.
- To characterize the functional impact of specific mutations on BR's photocycle.
- To explore the mechanism of light-induced photocurrent inversion.
Main Methods:
- Attachment of purple membranes to planar lipid bilayers.
- Measurement of pump currents via capacitive coupling and ionophores.
- Spectroscopic analysis of chromophore protonation states.
Main Results:
- All mutants displayed reduced Schiff base pK values (8-8.5).
- Continuous blue light induced outward proton pump currents.
- Azide significantly enhanced currents in D85N and D85,96N, but not D85T.
- Sequential yellow and blue light induced inverted photocurrents, similar to halorhodopsin.
Conclusions:
- The studied BR mutants exhibit altered proton translocation properties.
- Azide sensitivity suggests a modified proton access pathway in D85N and D85,96N.
- The observed photocurrent inversion is explained by retinal isomerization and differential proton accessibility.
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