Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation
Identifieur interne : 002D44 ( Main/Merge ); précédent : 002D43; suivant : 002D45Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation
Auteurs : Ji Luo [États-Unis] ; Rajendra Uprety [États-Unis] ; Yuta Naro [États-Unis] ; Chungjung Chou [États-Unis] ; Duy P. Nguyen [Royaume-Uni] ; Jason W. Chin [Royaume-Uni] ; Alexander Deiters [États-Unis]Source :
- Journal of the American Chemical Society [ 0002-7863 ] ; 2014.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Proteins.
- chemical , physiology : Proteins.
- chemical : Molecular Probes.
- chemistry : Methanosarcina barkeri.
- Chromatography, Liquid, Fluorescence, HEK293 Cells, Humans, Photons, Tandem Mass Spectrometry.
Abstract
The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.
Url:
DOI: 10.1021/ja5055862
PubMed: 25341086
PubMed Central: 4333581
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Encoded Optochemical Probes for Simultaneous
Fluorescence Reporting and Light Activation of Protein Function with
Two-Photon Excitation</title><author><name sortKey="Luo, Ji" sort="Luo, Ji" uniqKey="Luo J" first="Ji" last="Luo">Ji Luo</name><affiliation wicri:level="1"><nlm:aff id="aff1">Department of Chemistry,<institution>University of Pittsburgh</institution>, Pittsburgh, Pennsylvania 15260,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff2">Department of Chemistry,<institution>North Carolina State University</institution>, Raleigh, North Carolina 27695,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Uprety, Rajendra" sort="Uprety, Rajendra" uniqKey="Uprety R" first="Rajendra" last="Uprety">Rajendra Uprety</name><affiliation wicri:level="1"><nlm:aff id="aff2">Department of Chemistry,<institution>North Carolina State University</institution>, Raleigh, North Carolina 27695,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Naro, Yuta" sort="Naro, Yuta" uniqKey="Naro Y" first="Yuta" last="Naro">Yuta Naro</name><affiliation wicri:level="1"><nlm:aff id="aff1">Department of Chemistry,<institution>University of Pittsburgh</institution>, Pittsburgh, Pennsylvania 15260,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chou, Chungjung" sort="Chou, Chungjung" uniqKey="Chou C" first="Chungjung" last="Chou">Chungjung Chou</name><affiliation wicri:level="1"><nlm:aff id="aff2">Department of Chemistry,<institution>North Carolina State University</institution>, Raleigh, North Carolina 27695,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Nguyen, Duy P" sort="Nguyen, Duy P" uniqKey="Nguyen D" first="Duy P." last="Nguyen">Duy P. Nguyen</name><affiliation wicri:level="1"><nlm:aff id="aff3">Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH,<country>United Kingdom</country></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chin, Jason W" sort="Chin, Jason W" uniqKey="Chin J" first="Jason W." last="Chin">Jason W. Chin</name><affiliation wicri:level="1"><nlm:aff id="aff3">Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH,<country>United Kingdom</country></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Deiters, Alexander" sort="Deiters, Alexander" uniqKey="Deiters A" first="Alexander" last="Deiters">Alexander Deiters</name><affiliation wicri:level="1"><nlm:aff id="aff1">Department of Chemistry,<institution>University of Pittsburgh</institution>, Pittsburgh, Pennsylvania 15260,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Journal of the American Chemical Society</title><idno type="ISSN">0002-7863</idno><idno type="eISSN">1520-5126</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chromatography, Liquid</term><term>Fluorescence</term><term>HEK293 Cells</term><term>Humans</term><term>Methanosarcina barkeri (chemistry)</term><term>Molecular Probes</term><term>Photons</term><term>Proteins (chemistry)</term><term>Proteins (physiology)</term><term>Tandem Mass Spectrometry</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cellules HEK293</term><term>Chromatographie en phase liquide</term><term>Fluorescence</term><term>Humains</term><term>Methanosarcina barkeri ()</term><term>Photons</term><term>Protéines ()</term><term>Protéines (physiologie)</term><term>Sondes moléculaires</term><term>Spectrométrie de masse en tandem</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="physiology" xml:lang="en"><term>Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Molecular Probes</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Methanosarcina barkeri</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Protéines</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Chromatography, Liquid</term><term>Fluorescence</term><term>HEK293 Cells</term><term>Humans</term><term>Photons</term><term>Tandem Mass Spectrometry</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cellules HEK293</term><term>Chromatographie en phase liquide</term><term>Fluorescence</term><term>Humains</term><term>Methanosarcina barkeri</term><term>Photons</term><term>Protéines</term><term>Sondes moléculaires</term><term>Spectrométrie de masse en tandem</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ja-2014-055862_0006" id="ab-tgr1"></graphic></p><p>The site-specific
incorporation of three new coumarin lysine analogues
into proteins was achieved in bacterial and mammalian cells using
an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded
coumarin lysines were successfully applied as fluorescent cellular
probes for protein localization and for the optical activation of
protein function. As a proof-of-principle, photoregulation of firefly
luciferase was achieved in live cells by caging a key lysine residue,
and excellent OFF to ON light-switching ratios were observed. Furthermore,
two-photon and single-photon optochemical control of EGFP maturation
was demonstrated, enabling the use of different, potentially orthogonal
excitation wavelengths (365, 405, and 760 nm) for the sequential activation
of protein function in live cells. These results demonstrate that
coumarin lysines are a new and valuable class of optical probes that
can be used for the investigation and regulation of protein structure,
dynamics, function, and localization in live cells. The small size
of coumarin, the site-specific incorporation, the application as both
a light-activated caging group and as a fluorescent probe, and the
broad range of excitation wavelengths are advantageous over other
genetically encoded photocontrol systems and provide a precise and
multifunctional tool for cellular biology.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bort, G" uniqKey="Bort G">G. Bort</name></author><author><name sortKey="Gallavardin, T" uniqKey="Gallavardin T">T. Gallavardin</name></author><author><name sortKey="Ogden, D" uniqKey="Ogden D">D. Ogden</name></author><author><name sortKey="Dalko, P I" uniqKey="Dalko P">P. I. Dalko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Krueger, A T" uniqKey="Krueger A">A. T. Krueger</name></author><author><name sortKey="Imperiali, B" uniqKey="Imperiali B">B. Imperiali</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goncalves, M S" uniqKey="Goncalves M">M. S. Goncalves</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Klan, P" uniqKey="Klan P">P. Klan</name></author><author><name sortKey="Solomek, T" uniqKey="Solomek T">T. Solomek</name></author><author><name sortKey="Bochet, C G" uniqKey="Bochet C">C. G. Bochet</name></author><author><name sortKey="Blanc, A" uniqKey="Blanc A">A. Blanc</name></author><author><name sortKey="Givens, R" uniqKey="Givens R">R. Givens</name></author><author><name sortKey="Rubina, M" uniqKey="Rubina M">M. Rubina</name></author><author><name sortKey="Popik, V" uniqKey="Popik V">V. Popik</name></author><author><name sortKey="Kostikov, A" uniqKey="Kostikov A">A. Kostikov</name></author><author><name sortKey="Wirz, J" uniqKey="Wirz J">J. Wirz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brieke, C" uniqKey="Brieke C">C. Brieke</name></author><author><name sortKey="Rohrbach, F" uniqKey="Rohrbach F">F. Rohrbach</name></author><author><name sortKey="Gottschalk, A" uniqKey="Gottschalk A">A. Gottschalk</name></author><author><name sortKey="Mayer, G" uniqKey="Mayer G">G. Mayer</name></author><author><name sortKey="Heckel, A" uniqKey="Heckel A">A. Heckel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Riggsbee, C X0a W" uniqKey="Riggsbee C">C.
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