Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.
nanoscopy
photoswitchable fluorescent proteins
quantum chemistry
serial femtosecond crystallography
switching contrast
Journal
Chemphyschem : a European journal of chemical physics and physical chemistry
ISSN: 1439-7641
Titre abrégé: Chemphyschem
Pays: Germany
ID NLM: 100954211
Informations de publication
Date de publication:
06 10 2022
06 10 2022
Historique:
revised:
25
06
2022
received:
22
03
2022
pubmed:
13
8
2022
medline:
12
10
2022
entrez:
12
8
2022
Statut:
ppublish
Résumé
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
Identifiants
pubmed: 35959919
doi: 10.1002/cphc.202200192
doi:
Substances chimiques
Luminescent Proteins
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
e202200192Subventions
Organisme : U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences
ID : DE-AC02-76SF00515
Organisme : NIH HHS
ID : P41 GM103393
Pays : United States
Organisme : NIH HHS
ID : P41RR001209
Pays : United States
Organisme : Max Planck Society
Organisme : CNRS
ID : ANR-17-CE11-0047-01
Organisme : NIH HHS
ID : P41 GM103393
Pays : United States
Organisme : NIH HHS
ID : P41RR001209
Pays : United States
Informations de copyright
© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.
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