A pKa calculation of residues in a proton pump, bacteriorhodopsin, from structures determined by electron crystallography

Bacteriorhodopsin (bR) is a light-driven proton pump, which is a membrane protein found in halophilic archeae like Halobacterium salinarum and in eubacteria [1]. When the covalently bound retinal chromophore absorbs the light energy, it changes the conformation from all-trans to 13-cis. This configuration change initiates ion translocation across the cell membrane and a proton moves from inside to outside of the cell. The bR molecules are forming two-dimensional crystals on the membranes of halophilic archeae, and therefore the atomic model of bR was first determined by electron crystallography. The determined structure can be used to determine the pKa values, through which the charge states of ionizable residues in bR determine their pH-dependent properties. The pH-dependent properties are crucial for proton translocation from ionizable residues or to ionizable residues. Detection of the intermediate states of the reaction cycle (photocycle) produced spectroscopic information, which can predict the ionization state of the ionozable residues. In the transition from the L intermediate to the M intermediate, it is known that a proton moves from the Shiff base on the retinal chromophore to Asp85, while a proton is released to the extracellar side from proton-releasing groups including Glu194 and Glu204. Experimentally the pKa value of the proton release is determined to be about 9.7, while the pKa value of Asp85 was measured to change from 2.6 to 7.5 by the proton release from the proton-releasing groups [2]. Here we used the PROPKA program [3] to calculate the pKa values of Asp85 and the proton-releasing groups from the structures at pH 5.5 and at pH 10.0 determined by electron crystallography. The calculation showed that the pKa value of Asp85 changes from 5.3 to 6.1, which qualitatively show the similar changes with the measured difference. The largest change between the structures is the shift of Arg82 by the proton release from the proton-releasing groups. However, the calculation showed that the electrostatic change from the shift cannot explain the pKa change of Asp85. The calculation indicated that the breakage of a hydrogen bond between Thr89 and Asp85, which is resulted from the backbone shift caused by the side chain shift of Arg82, is the main cause of the pKa change. In addition, the structures showed the shift of a water molecule near Asp85, which could enhance the pKa change on Asp85. The pKa calculation using the structures determined by electron crystallography could be a valuable tool to understand the functions of proteins.