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Asp96 deprotonation and transmembrane alpha-helical structural changes in bacteriorhodopsin
K J Rothschild1, T Marti, S Sonar
1Physics Department, Boston University, Massachusetts 02215.
The Journal of Biological Chemistry
|December 25, 1993
Summary
Replacing Thr46 with Aspartic acid in bacteriorhodopsin accelerates N formation and slows N decay. This mutation facilitates studying the N intermediate and reveals Asp96
Area of Science:
- Biophysics
- Structural Biology
- Spectroscopy
Background:
- The M to N transition in bacteriorhodopsin involves proton transfer and conformational changes.
- The role of specific residues, like Thr46, in this proton transfer network is not fully understood.
Purpose of the Study:
- To investigate the role of Thr46 in the M-->N transition of bacteriorhodopsin.
- To characterize the effects of a T46D mutation on the photocycle kinetics and intermediate formation.
Main Methods:
- Fourier transform infrared (FTIR) spectroscopy
- Resonance Raman spectroscopy
- Site-directed mutagenesis (T46D mutant)
Main Results:
- The T46D mutant exhibits normal chromophore structure but altered photocycle kinetics.
- N intermediate formation is accelerated, and N decay is slowed in the T46D mutant, leading to accumulation.
- FTIR difference spectroscopy shows altered Asp96 environment and a reduced pKa in the T46D mutant.
Conclusions:
- The T46D mutation modulates the pKa of Asp96, impacting photocycle kinetics.
- Backbone structural changes, involving transmembrane alpha-helices, influence Asp96's protonation state.
- These changes are crucial for proton transfer and the formation of the hydrogen-bonded network during Schiff base reprotonation.