Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins

A thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in the Faculty of Physics and Materials Science.

Detalhes bibliográficos
Autor: Torcal Embeita, Bruno
Formato: tesis doctoral
Estado:Versión publicada
Fecha de publicación:2016
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/180226
Acesso em linha:http://hdl.handle.net/10261/180226
Access Level:acceso abierto
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spelling Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteinsTorcal Embeita, BrunoA thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in the Faculty of Physics and Materials Science.Reversibly photoswitchable uorescent proteins (RSFPs) are genetically engineered proteins that can be switched by light absorption between a uorescent ON state and a dark OFF state. Among other applications they allow to increase the resolution beyond the difraction limit in cell imaging by fluorescence microscopy. RSFPs have extended the possibilities of fluorescence microscopy and other biotechnological tools, but the development of their properties is still far from being rationally designed. Thus, there might be much room for improvement if we manage to understand the switching mechanisms. The switching mechanisms in several negative RSFPs is being elucidated but still under debate. In this thesis I extend the theoretical knowledge about the photoswitching of negative RSFPs by studying the excited-state potential energy surface of both the ON- and the OFF-state. I compare three RSFPs, namely IrisFP, Dronpa and a fast switching single mutant of Dronpa called Dronpa2 to search for the origin of their different switching quantum yields in the ON-state. For the OFF-state, the results of the combined quantum mechanics-molecular mechanics (QM/MM) calculations show that chromophore photoisomerization happens in its neutral form and is followed by ground state deprotonation. This is in agreement with a very recent ultrafast absorption spectroscopy study for IrisFP, and studies on Dronpa and other negative RSFPs. Although the experimental results in both proteins show the same steps, I found that they have different processes at the atomic level due to structural and electrostatical differences, but leading to the same intermediates. This is in contrast to the ON-state where I get the same picture for the three proteins studied, identifying the conical intersection that quenches the uorescence and controls the photoswitching quantum yield. The major difference between the three proteins in terms of fluorescence and photoswitching characteristics comes from the different sterical environment produced by the residue 159, which is a methionine in different isomers in IrisFP and Dronpa and a smaller threonine that allows a faster isomerization of the chromophore in Dronpa2.Peer reviewedUniversidad del País VascoWanko, MariusRubio, AngelConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192016info:eu-repo/semantics/doctoralThesishttp://purl.org/coar/resource_type/c_db06Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/180226reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1802262026-05-22T06:33:51Z
dc.title.none.fl_str_mv Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
title Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
spellingShingle Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
Torcal Embeita, Bruno
title_short Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
title_full Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
title_fullStr Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
title_full_unstemmed Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
title_sort Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
dc.creator.none.fl_str_mv Torcal Embeita, Bruno
author Torcal Embeita, Bruno
author_facet Torcal Embeita, Bruno
author_role author
dc.contributor.none.fl_str_mv Wanko, Marius
Rubio, Angel
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
description A thesis submitted in partial fulfillment for the degree of Doctor of Philosophy in the Faculty of Physics and Materials Science.
publishDate 2016
dc.date.none.fl_str_mv 2016
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
http://purl.org/coar/resource_type/c_db06
Publisher's version
info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/180226
url http://hdl.handle.net/10261/180226
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Universidad del País Vasco
publisher.none.fl_str_mv Universidad del País Vasco
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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