A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.
cyanobacteriochromes
fluorescence
fluorescent proteins
phycoerythrobilin
protein engineering
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
04 10 2023
04 10 2023
Historique:
revised:
06
07
2023
received:
11
05
2023
pmc-release:
04
10
2024
medline:
11
10
2023
pubmed:
10
7
2023
entrez:
9
7
2023
Statut:
ppublish
Résumé
Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.
Identifiants
pubmed: 37423892
doi: 10.1002/cbic.202300358
pmc: PMC10653908
mid: NIHMS1936845
doi:
Substances chimiques
Adenylyl Cyclases
EC 4.6.1.1
Bacterial Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
e202300358Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM138223
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
© 2023 Wiley-VCH GmbH.
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