Spectroscopic Characterization of Atypical Ion Pumping Microbial Rhodopsins

Microbial rhodopsins are seven transmembrane α-helical proteins with a retinal cofactor which affords the sensitivity to a broad spectrum of visible light and provides the driving energy needed for transport. They are ubiquitous in nature, are expressed in all three domains of life, and their hosts are found in a wide variety of environments. A great deal is known regarding proton transport in the extracellular direction and chloride transport in the cytoplasmic direction, primarily through the study of the prototypical proton pump, bacteriorhodopsin and archaeal chloride pumping halorhodopsins. Recently, in the last six years, microbial rhodopsin chloride transport in bacteria and inward proton transport were discovered. Here, two new and atypical groups of microbial rhodopsin ion pumps are described and characterized largely through time-resolved vibrational and visible light spectroscopy in parallel with site directed mutagenesis. First, we investigated a group of chloride pumping microbial rhodopsins from cyanobacteria with an unusual, bacteriorhodopsin-like sequence. We observed deprotonation of a key residue in the so-called proton donor position, which is likely a regulatory mechanism to ensure efficient chloride transport and prevent the backflow of chloride. Next, a new group of inward proton pumps from Antarctic, alkaline, freshwater lakes with a highly hydrophilic primary sequence was characterized. The mechanism of proton transport, along with several important residues were identified including a central amino acid which forms hydrogen bonds to bridge the cytoplasmic and extracellular sides of the protein and is apparently crucial for transport. Additionally, two thermally stable isomeric forms of retinal are accommodated in the binding pocket and the equilibrium between these states is dependent on the wavelength of illumination and pH. Hypotheses regarding the unanswered question of the biological role of inward proton transport are discussed. Ion transporting microbial rhodopsins are of interest to researchers in the field of optogenetics for the optical control of cells or organelles that express them. These proteins have the potential to be used in the field in their native state or with improved properties after mutagenesis.