Absorption, fluorescence and excitation spectra of three-dimensional
bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct
protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing
bacteriorhodopsin, we suggest that
amino acid residues, located in the vicinity of the
retinal chromophore, have changed their protonation state. We propose partial
dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional
dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated
bacteriorhodopsin membranes. Full hydration of the
proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by
lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from
proteins in different protonation states.