Spectral tuning of Archaerhodopsin 3 in Escherichia coli
<p>Microbial rhodopsins are proteins that act as light driven proton pumps located in the plasma membrane of eukarya, bacteria and archaea. These proteins facilitate the movement of protons by light-induced isomerisation of a central retinal chromophore found in the transmembrane core of the o...
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| Formato: | Thesis |
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2018
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| _version_ | 1826285636501372928 |
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| author | Crowther, O |
| author2 | Watts, A |
| author_facet | Watts, A Crowther, O |
| author_sort | Crowther, O |
| collection | OXFORD |
| description | <p>Microbial rhodopsins are proteins that act as light driven proton pumps located in the plasma membrane of eukarya, bacteria and archaea. These proteins facilitate the movement of protons by light-induced isomerisation of a central retinal chromophore found in the transmembrane core of the opsin protein. Here we detail an extensive study of amino acid substitutions in the retinal binding pocket of archaerhodopsin 3 (AR3) and their effect on the absorbance spectrum of the protein for use in biotechnological applications.</p> <p>In this thesis, protein engineering was used to generate a mutant library that produced hypsochromic and bathochromic shifts when purified from an E. coli model into a detergent environment. The largest shifts reported here (-25 and +7 nm compared to wtAR3) were achieved through strategic mutation of residues interacting with the retinal chromophore. The S151C mutant produced in this report perturbed the photocycle kinetics in numerous detergent systems tested, resulting in a delayed photo-intermediate decay and return to ground state.</p> <p>The faster photocycle kinetics of AR3 compared to other rhodopsins makes it a promising target for both optogenetic and bio-sensory applications. For its use as a single molecule bio-sensor, a strategic cysteine mutation was incorporated into the intracellular EF loop of the protein to allow for its future immobilisation onto a conductive surface. With monomeric AR3 adsorbed onto a gold substrate, it can be assessed for its ability to generate a detectable proton current in response to activation by light of a wavelength specific to each mutant. For applications in optogenetics, distinctly shifted mutants can allow for targeted activation of different mutants in the same neural system, thus allowing for the assaying of more complex pathways. Red-shifted mutants are of particular interest in this field as they allow for non-invasive light penetration in the brain.</p> |
| first_indexed | 2024-03-07T01:31:48Z |
| format | Thesis |
| id | oxford-uuid:93d98317-191d-435f-b687-df188d6e05dd |
| institution | University of Oxford |
| last_indexed | 2024-03-07T01:31:48Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | oxford-uuid:93d98317-191d-435f-b687-df188d6e05dd2022-03-26T23:35:10ZSpectral tuning of Archaerhodopsin 3 in Escherichia coliThesishttp://purl.org/coar/resource_type/c_bdccuuid:93d98317-191d-435f-b687-df188d6e05ddORA Deposit2018Crowther, OWatts, A<p>Microbial rhodopsins are proteins that act as light driven proton pumps located in the plasma membrane of eukarya, bacteria and archaea. These proteins facilitate the movement of protons by light-induced isomerisation of a central retinal chromophore found in the transmembrane core of the opsin protein. Here we detail an extensive study of amino acid substitutions in the retinal binding pocket of archaerhodopsin 3 (AR3) and their effect on the absorbance spectrum of the protein for use in biotechnological applications.</p> <p>In this thesis, protein engineering was used to generate a mutant library that produced hypsochromic and bathochromic shifts when purified from an E. coli model into a detergent environment. The largest shifts reported here (-25 and +7 nm compared to wtAR3) were achieved through strategic mutation of residues interacting with the retinal chromophore. The S151C mutant produced in this report perturbed the photocycle kinetics in numerous detergent systems tested, resulting in a delayed photo-intermediate decay and return to ground state.</p> <p>The faster photocycle kinetics of AR3 compared to other rhodopsins makes it a promising target for both optogenetic and bio-sensory applications. For its use as a single molecule bio-sensor, a strategic cysteine mutation was incorporated into the intracellular EF loop of the protein to allow for its future immobilisation onto a conductive surface. With monomeric AR3 adsorbed onto a gold substrate, it can be assessed for its ability to generate a detectable proton current in response to activation by light of a wavelength specific to each mutant. For applications in optogenetics, distinctly shifted mutants can allow for targeted activation of different mutants in the same neural system, thus allowing for the assaying of more complex pathways. Red-shifted mutants are of particular interest in this field as they allow for non-invasive light penetration in the brain.</p> |
| spellingShingle | Crowther, O Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title | Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title_full | Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title_fullStr | Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title_full_unstemmed | Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title_short | Spectral tuning of Archaerhodopsin 3 in Escherichia coli |
| title_sort | spectral tuning of archaerhodopsin 3 in escherichia coli |
| work_keys_str_mv | AT crowthero spectraltuningofarchaerhodopsin3inescherichiacoli |