The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.
X-ray crystallography
flavoprotein
fluorescence
imaging
photophysics
rational protein design
spectral tuning
structure–function flavin-binding fluorescent protein
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
Historique:
received:
07
01
2021
revised:
09
04
2021
accepted:
12
04
2021
pubmed:
17
4
2021
medline:
24
8
2021
entrez:
16
4
2021
Statut:
ppublish
Résumé
Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.
Identifiants
pubmed: 33862085
pii: S0021-9258(21)00450-6
doi: 10.1016/j.jbc.2021.100662
pmc: PMC8131319
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Flavins
0
Luminescent Proteins
0
Mutant Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
100662Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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