3CD9

Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments

3CD9 の概要

• エントリーDOI: 10.2210/pdb3cd9/pdb
• 関連するPDBエントリー: 3CB9 3CBE 3CD1
• 分子名称: Green fluorescent protein (2 entities in total)
• 機能のキーワード: gfp, redox, luminescent protein
• 由来する生物種: Aequorea victoria (Jellyfish)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 28138.56

構造登録者

Lohman, J.R. (登録日: 2008-02-26, 公開日: 2008-08-19, 最終更新日: 2024-11-06)

主引用文献

Lohman, J.R.,Remington, S.J.
Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments
Biochemistry, 47:8678-8688, 2008

PubMed Abstract: Green fluorescent protein (GFP) indicators were previously developed that rapidly and quantitatively respond to changes in the thiol/disulfide equilibrium within subcellular compartments. In these indicators, surface-exposed cysteines residues were introduced so as to form a labile redox-active disulfide that in turn controls the emission properties of the internal chromophore. The biosensors have been shown to be effective reporters of the thiol/disulfide status within reducing compartments such as the mitochondria and cytosol for several cell types. However, due to the high thermodynamic stability of the introduced disulfide bond, the indicators are not useful for quantitative analysis within more oxidizing compartments such as the endoplasmic reticulum. Here we report the development of a new family of GFP-based redox indicators (roGFP1-iX) in which the thermodynamic stability of the disulfide is substantially lowered by insertion of a single amino acid into the main chain, adjacent to cysteine 147. The insertions result in indicators with midpoint potentials of -229 to -246 mV and are thus better suited for study of relatively oxidizing subcellular compartments. Atomic resolution crystallographic analyses suggest that two important factors act to destabilize the disulfide linkage in roGFP1-iX. In the oxidized state, an unusual non-proline cis-peptide bond adjacent to one of the cysteines introduces geometric strain into the system, while in the reduced state, a dramatic loop opening lowers the effective concentration of the reacting species.

PubMed: 18652491
DOI: 10.1021/bi800498g

実験手法

X-RAY DIFFRACTION (1.502 Å)