pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy

Area of Science:

• Biochemistry
• Spectroscopy
• Protein structure

Background:

• Bacteriorhodopsin (bR) contains four internal aspartic acid residues.
• Previous studies indicated protonation of Asp-96 and Asp-115 up to pH 10.
• Accurate pKa values for these residues were not previously determined.

Purpose of the Study:

• To characterize pH-dependent structural changes in wild-type bacteriorhodopsin and its D96N mutant.
• To determine the pKa values of internal aspartic acid residues.
• To elucidate the role of Asp-96 in protein conformational changes.

Main Methods:

• Infrared spectroscopy utilizing the attenuated total reflection technique.
• Analysis of pH-dependent spectral changes in ground-state, dark-adapted wild-type bR.
• Comparison of spectral data between wild-type bR and the D96N mutant.

Main Results:

• Deprotonation of Asp-96 observed at high pH with a pKa of 11.4 ± 0.1.
• No direct titration of Asp-115 was detected.
• Complex conformational changes revealed additional pKa values (pKa1 = 9.3 ± 0.3, pKa2 = 11.5 ± 0.2).
• The pKa at ~11.5 reflects a conformational change affecting Asp-96 accessibility, not direct titration.

Conclusions:

• Asp-96 deprotonation occurs at a high pKa of 11.4.
• A distinct protein conformational change, occurring at pKa ~11.5, influences Asp-96's accessibility.
• Asp-115 titration was not observed under the experimental conditions.