Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.
FEBS J
; 286(12): 2329-2340, 2019 06.
Article
em En
| MEDLINE
| ID: mdl-30817081
ABSTRACT
Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7 Å resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Conformação Proteica
/
Dobramento de Proteína
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Proteínas de Fluorescência Verde
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Aminoácidos
Idioma:
En
Revista:
FEBS J
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Alemanha
País de publicação:
ENGLAND
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ESCOCIA
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GB
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GREAT BRITAIN
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INGLATERRA
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REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM