Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

Abstract

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

Progress in pH-Sensitive sensors: essential tools for organelle pH detection, spotlighting mitochondrion and diverse applications

“…The paragraph also reports the three-dimensional structures of both the native green and photoconverted red forms of DendFP. Additionally, these findings highlight the role of specific amino acid positions, such as Ser142 and His193, in influencing the photoconversion rate, emphasizing the importance of hydrogen bonding between the chromophore and the Gln116 and Ser105 clusters for this process ( Pletneva et al, 2016 ).…”

Definition of “Hotspots” to Improve the Maturation of the Fluorescent Protein Moxsaasoti at 37 °c

“…Такие белки нашли широкое применение в качестве меток для методов микроскопии сверхвысокого разрешения, позволяя визуализировать структуры за пределами дифракционного барьера [4][5][6]. К таким белкам относятся фотоконвертируемые флуоресцентные белки (ФКФБ), способные менять длину волны эмиссии (сине-зеленые, оранжево-красные, и наиболее распространенные благодаря своей контрастности -зелено-красные ФКФБ [7][8][9][10][11]), фотопереключаемые белки (ФПФБ), способные переключаться между флуоресцентным и нефлуоресцентным состояниями [12][13][14][15][16]. Также существует малочисленная группа бифотохромных флуоресцентных белков, объединяющих в себе предыдущие два свойства, что позволяет использовать их в комбинации методов суперразрешающей микроскопии и получать более точные изображения [17].…”

Fluorescent reporters, calibration standards and endogenous protein labeling for quantitative single-molecule localization microscopy in cells

“…In particular, the residue 69 could be of importance, which differs in mEos3.2 (alanine) and mMaple3 (threonine) and which is assumed to have a catalytic function in the formation of the red chromophore (Stepanenko et al 2013). Furthermore, this residue is in close proximity to the amino acids His62-Tyr63-Gly64, which constitute the chromophore, and residue 69 could possibly influence the stability of the chromophore (Pletneva et al 2016;Bourgeois 2017).…”