5BT0

Switching GFP fluorescence using genetically encoded phenyl azide chemistry through two different non-native post-translational modifications routes at the same position.

5BT0 の概要

• エントリーDOI: 10.2210/pdb5bt0/pdb
• 分子名称: Green fluorescent protein, SULFATE ION (3 entities in total)
• 機能のキーワード: synthetic biology, photocontrol, optogenetics, unnatural amino acids, protein fluorescence, sfgfp, fluorescent protein
• 由来する生物種: Aequorea victoria (Jellyfish)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 52891.21

構造登録者

Hartley, A.M.,Worthy, H.L.,Reddington, S.C.,Rizkallah, P.J.,Jones, D.D. (登録日: 2015-06-02, 公開日: 2016-07-13, 最終更新日: 2017-05-10)

主引用文献

Hartley, A.M.,Worthy, H.L.,Reddington, S.C.,Rizkallah, P.J.,Jones, D.D.
Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle.
Chem Sci, 7:6484-6491, 2016

PubMed Abstract: Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function and either photochemistry or bioconjugation. Replacement of H148 with -azido-l-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and original protein. Crystal structures of both the click modified and photochemically converted forms show that functional switching is due to local conformational changes that optimise the interaction networks surrounding the chromophore. Crystal structure and mass spectrometry studies of the irradiated protein suggest that the phenyl azide converts to a dehydroazepine and/or an azepinone. Thus, protein embedded phenyl azides can be used beyond simple photocrosslinkers and passive conjugation handles, and mimic many natural post-translational modifications: modulation though changes in interaction networks.

PubMed: 28451106
DOI: 10.1039/c6sc00944a

実験手法

X-RAY DIFFRACTION (2.03 Å)