Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Bacteriorhodopsin is a light-driven proton pump found in purple membranes.
• Crystallization of membrane proteins can alter their native state and properties.

Purpose of the Study:

• To investigate the heterogeneity of bacteriorhodopsin within three-dimensional crystals.
• To understand the impact of crystallization conditions on bacteriorhodopsin's protonation states and hydration.

Main Methods:

• Absorption, fluorescence, and excitation spectroscopy at room temperature, controlled pH, and full external hydration.
• Comparison with spectra from dehydrated and deionized membranes.
• Crystallographic data analysis.

Main Results:

• Identification of three distinct bacteriorhodopsin species, with two novel forms comprising up to 30% of the total.
• Spectra suggest altered protonation states of amino acid residues near the retinal chromophore.
• Interconversion of species observed upon dehydration, supported by crystallographic data showing similarity to dehydrated membranes.
• Hindered full hydration post-crystallization attributed to lipid bilayer closure of hydrophobic surfaces.

Conclusions:

• Crystallization conditions, particularly partial dehydration, induce heterogeneity in bacteriorhodopsin protonation states.
• Spectroscopic methods quantitatively reveal compositional heterogeneity, complementing crystallographic findings.
• Understanding this heterogeneity is crucial for interpreting bacteriorhodopsin structure-function relationships in crystalline states.