X-ray absorption and molecular dynamics study of cation binding sites in the purple membrane

The present work describes the results of a study aimed at identifying candidate cation binding sites on the extracellular region of bacteriorhodopsin, including a site near the retinal pocket. The approach used is a combined effort involving computational chemistry methods (computation of cation affinity maps and molecular dynamics) together with the Extended X‐Ray Absorption Fine Structure (EXAFS) technique to obtain relevant information about the local structure of the protein in the neighborhood of Mn2+ ions in different affinity binding sites. The results permit the identification of a high‐affinity binding site where the ion is coordinated simultaneously to Asp212− and Asp85−. Comparison of EXAFS data of the wild type protein with the quadruple mutant E9Q/E74Q/E194Q/E204Q at pH 7.0 and 10.0 demonstrate that extracellular glutamic acid residues are involved in cation binding. Proteins 2007 © 2007 Wiley‐Liss, Inc.