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

Abstract

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 (UnaG^Y99F_Y134W, UnaG^N57E, UnaG^L41F, and UnaG^F17M) 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, UnaG^L41F has the highest affinity and stability among the mutants.

Keywords:

• Bilirubin
• fluorescence protein
• UnaG

Acknowledgments

This study was supported by The Scientific and Technological Research Council of Turkey (Grant Number 215Z700) and British Council of UK (Grant Number RES/0120/7785). We also thank Helen Waller and Daniel T. Peters. This study was partially produced from Numan Eczacioglu Ph.D. thesis.

Disclosure statement

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human or animal subjects performed by any of the authors. We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal.