Spectroscopic Characterization of Atypical Ion Pumping Microbial Rhodopsins

dc.contributor.advisorBrown, Leonid
dc.contributor.authorHarris, Andrew
dc.date.accessioned2020-09-17T16:52:00Z
dc.date.available2020-09-17T16:52:00Z
dc.date.copyright2020-09
dc.date.created2020-09-03
dc.degree.departmentDepartment of Physicsen_US
dc.degree.grantorUniversity of Guelphen_US
dc.degree.nameDoctor of Philosophyen_US
dc.degree.programmeBiophysicsen_US
dc.description.abstractMicrobial 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.en_US
dc.description.sponsorshipNatural Sciences and Engineering Research Council of Canada
dc.description.sponsorshipOGS
dc.description.sponsorshipUniversity of Guelph
dc.identifier.urihttps://hdl.handle.net/10214/21298
dc.language.isoenen_US
dc.publisherUniversity of Guelphen_US
dc.rightsAttribution 4.0 International*
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/*
dc.subjectrhodopsinen_US
dc.subjectftiren_US
dc.subjectspectroscopyen_US
dc.subjection translocationen_US
dc.subjectoptogeneticsen_US
dc.subjectmicrobial rhodopsinen_US
dc.subjectflash photolysisen_US
dc.subjectmolecular mechanismen_US
dc.subjectchloride transporten_US
dc.subjectinward proton transporten_US
dc.subjectbistableen_US
dc.titleSpectroscopic Characterization of Atypical Ion Pumping Microbial Rhodopsinsen_US
dc.typeThesisen_US

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