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Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties

Lookup NU author(s): Numan Eczacioglu, Dr Yakup Ulusu, Dr Isa Gokce, Professor Jeremy LakeyORCiD

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Abstract

© 2021 Taylor & Francis Group, LLC. The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Publication metadata

Author(s): Eczacioglu N, Ulusu Y, Gokce I, Lakey JH

Publication type: Article

Publication status: Published

Journal: Preparative Biochemistry and Biotechnology

Year: 2022

Volume: 52

Issue: 4

Pages: 365-374

Online publication date: 28/07/2021

Acceptance date: 02/04/2018

ISSN (print): 1082-6068

ISSN (electronic): 1532-2297

Publisher: Taylor and Francis Ltd

URL: https://doi.org/10.1080/10826068.2021.1952597

DOI: 10.1080/10826068.2021.1952597

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