8ARM

Crystal structure of LSSmScarlet2

8ARM の概要

• エントリーDOI: 10.2210/pdb8arm/pdb
• 分子名称: LSSmScarlet2, SULFATE ION (3 entities in total)
• 機能のキーワード: fluorescent protein, lssmscarlet2
• 由来する生物種: Discosoma sp.
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 26610.04

構造登録者

Samygina, V.R.,Subach, O.M.,Vlaskina, A.V.,Subach, F.V. (登録日: 2022-08-17, 公開日: 2022-11-02, 最終更新日: 2024-01-31)

主引用文献

Subach, O.M.,Vlaskina, A.V.,Agapova, Y.K.,Piatkevich, K.D.,Patrushev, M.V.,Samygina, V.R.,Subach, F.V.
LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes' Shift.
Int J Mol Sci, 23:-, 2022

PubMed Abstract: Red fluorescent proteins with a large Stokes' shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes' shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein.

PubMed: 36232354
DOI: 10.3390/ijms231911051

実験手法

X-RAY DIFFRACTION (1.41 Å)