Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Bacteriorhodopsin is a light-driven proton pump.
• The Schiff base is crucial for proton transfer in bacteriorhodopsin.
• Mutations can alter protein function and proton binding.

Purpose of the Study:

• To investigate the effect of external electric fields on Asp-85-->Asn mutant bacteriorhodopsin.
• To compare the field-induced changes in the mutant to wild-type bacteriorhodopsin.
• To understand the role of Schiff base proton binding affinity in field response.

Main Methods:

• Preparation of dry films of Asp-85-->Asn mutant and wild-type bacteriorhodopsin.
• Application of external electric fields to the protein films.
• Optical absorption spectroscopy to monitor spectral shifts (e.g., 600 nm to 400 nm).
• Determination of Schiff base proton binding affinity (pKa).

Main Results:

• Electric field application to Asp-85-->Asn mutant films caused Schiff base deprotonation and a spectral shift from 600 nm to 400 nm.
• Wild-type bacteriorhodopsin films showed a red-shifted product under electric fields, distinct from the mutant.
• The mutant protein exhibited a significantly lower Schiff base proton pKa (~9) compared to wild-type (~13), indicating weaker proton binding.

Conclusions:

• The weaker Schiff base proton binding in the Asp-85-->Asn mutant is responsible for the field-induced deprotonation and spectral shift.
• Bacteriorhodopsins with reduced Schiff base pKa are predicted to show similar field-induced protonation equilibrium shifts.
• Proposed mechanisms explain the observed differences in field response between mutant and wild-type bacteriorhodopsin.