Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-8640
Main Title: The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP
Author(s): Buhrke, David
Vélazquez Escobar, Francisco
Sauthof, Luisa
Wilkening, Svea
Herder, Nico
Tavraz, Neslihan N.
Willoweit, Mario
Keidel, Anke
Utesch, Tillmann
Mroginski, Maria-Andrea
Schmitt, Franz-Josef
Hildebrandt, Peter
Friedrich, Thomas
Type: Article
Is Part Of: 10.14279/depositonce-8309
Language Code: en
Abstract: Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.
URI: https://depositonce.tu-berlin.de/handle/11303/9595
http://dx.doi.org/10.14279/depositonce-8640
Issue Date: 22-Jun-2016
Date Available: 5-Jul-2019
DDC Class: 540 Chemie und zugeordnete Wissenschaften
Subject(s): amino acid substitution
iRFP
bacteriophytochromes
protein
fluorescence
Sponsor/Funder: DFG, 221545957, SFB 1078: Proteinfunktion durch Protonierungsdynamik
DFG, 53182490, EXC 314: Unifying Concepts in Catalysis
License: https://creativecommons.org/licenses/by/4.0/
Journal Title: Scientific Reports
Publisher: Nature Publishing Group
Publisher Place: London
Volume: 6
Article Number: 28444
Publisher DOI: 10.1038/srep28444
EISSN: 2045-2322
Appears in Collections:FG Bioenergetik » Publications

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