RESUMO
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.
Assuntos
Aminoácidos/genética , Proteínas de Fluorescência Verde/química , Conformação Proteica , Dobramento de Proteína , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Methanocaldococcus/genética , Imagem Molecular/métodos , Mutação/genética , Fenilalanina/química , RNA de Transferência/química , RNA de Transferência/genética , Tirosina/genéticaRESUMO
We report a new synthetic route to a series of α-carboxynitrobenzyl photocaged l-aspartates for application in time-resolved structural biology. The resulting compounds were characterised in terms of UV/Vis absorption properties, aqueous solubility and stability, and photocleavage rates (τ = µs to ms) and quantum yields (φ = 0.05 to 0.14).