6H01

Crystal structure of a domain-swapped dark-state sfGFP containing the unnatural amino acid ortho-nitrobenzyl-tyrosine (ONBY) at residue 66

6H01 の概要

• エントリーDOI: 10.2210/pdb6h01/pdb
• 分子名称: Green fluorescent protein (2 entities in total)
• 機能のキーワード: gfp, unnatural amino acids, domain-swapped, fluorescent protein
• 由来する生物種: Aequorea victoria (Jellyfish)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 119633.65

構造登録者

Kesgin-Schaefer, S.,Tidow, H. (登録日: 2018-07-06, 公開日: 2019-04-24, 最終更新日: 2024-11-20)

主引用文献

Kesgin-Schaefer, S.,Heidemann, J.,Puchert, A.,Koelbel, K.,Yorke, B.A.,Huse, N.,Pearson, A.R.,Uetrecht, C.,Tidow, H.
Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.
Febs J., 286:2329-2340, 2019

PubMed 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.

PubMed: 30817081
DOI: 10.1111/febs.14797

実験手法

X-RAY DIFFRACTION (2.776 Å)