Specific binding sites for cations in bacteriorhodopsin

Area of Science:

• Biophysics
• Structural Biology
• Membrane Proteins

Background:

• Bacteriorhodopsin (bR) functions as a light-driven proton pump, with Asp-85 residue critical for its function.
• Protonation of Asp-85 induces a color transition in bR (purple to blue), a process also triggered by cation removal.
• Previous hypotheses suggested cations bind to the bR surface or internal protein sites, affecting surface pH or retinal vicinity.

Purpose of the Study:

• To reexamine cation binding mechanisms in bacteriorhodopsin.
• To determine the location of cation binding sites using electron paramagnetic resonance (EPR) spectroscopy.
• To elucidate the role of specific cation binding in modulating bR's spectral properties.

Main Methods:

• Utilized electron paramagnetic resonance (EPR) spectroscopy to study interactions between Mn(2+) and nitroxyl radical probes.
• Employed site-directed mutagenesis to introduce cysteine residues for covalent attachment of spin labels.
• Assessed the effect of Mn(2+) binding on EPR spectra of spin labels at specific positions (74C, 103C, 163C).

Main Results:

• Mn(2+) binding to the highest-affinity site significantly altered the EPR spectrum of a spin label at residue 74C (extracellular side).
• The distance between the high-affinity binding site and residue 74C was estimated at approximately 9.8 +/- 0.7 Å.
• Mn(2+) binding to low-affinity sites affected spin labels at residues 103C and 163C (cytoplasmic side), indicating cytoplasmic binding.

Conclusions:

• Identified specific cation binding sites on both the extracellular and cytoplasmic sides of bacteriorhodopsin.
• The highest-affinity cation binding site is located on the extracellular side, near residue 74C.
• Low-affinity cation binding sites are at least partially on the cytoplasmic side, suggesting involvement of negatively charged lipids on the bR exterior.