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Photophysics of the red-form Kaede chromophore

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, fac...

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Autores principales: Addison, Kiri, Roy, Palas, Bressan, Giovanni, Skudaite, Karolina, Robb, Josh, Bulman Page, Philip C., Ashworth, Eleanor K., Bull, James N., Meech, Stephen R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10074405/
https://www.ncbi.nlm.nih.gov/pubmed/37035701
http://dx.doi.org/10.1039/d3sc00368j
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author Addison, Kiri
Roy, Palas
Bressan, Giovanni
Skudaite, Karolina
Robb, Josh
Bulman Page, Philip C.
Ashworth, Eleanor K.
Bull, James N.
Meech, Stephen R.
author_facet Addison, Kiri
Roy, Palas
Bressan, Giovanni
Skudaite, Karolina
Robb, Josh
Bulman Page, Philip C.
Ashworth, Eleanor K.
Bull, James N.
Meech, Stephen R.
author_sort Addison, Kiri
collection PubMed
description The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.
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spelling pubmed-100744052023-04-06 Photophysics of the red-form Kaede chromophore Addison, Kiri Roy, Palas Bressan, Giovanni Skudaite, Karolina Robb, Josh Bulman Page, Philip C. Ashworth, Eleanor K. Bull, James N. Meech, Stephen R. Chem Sci Chemistry The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima. The Royal Society of Chemistry 2023-03-09 /pmc/articles/PMC10074405/ /pubmed/37035701 http://dx.doi.org/10.1039/d3sc00368j Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Addison, Kiri
Roy, Palas
Bressan, Giovanni
Skudaite, Karolina
Robb, Josh
Bulman Page, Philip C.
Ashworth, Eleanor K.
Bull, James N.
Meech, Stephen R.
Photophysics of the red-form Kaede chromophore
title Photophysics of the red-form Kaede chromophore
title_full Photophysics of the red-form Kaede chromophore
title_fullStr Photophysics of the red-form Kaede chromophore
title_full_unstemmed Photophysics of the red-form Kaede chromophore
title_short Photophysics of the red-form Kaede chromophore
title_sort photophysics of the red-form kaede chromophore
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10074405/
https://www.ncbi.nlm.nih.gov/pubmed/37035701
http://dx.doi.org/10.1039/d3sc00368j
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