Poster W12 - Characterizing the Binding Affinity of Newly-Discovered β-Hydroxybutyrate Binding Proteins for the Development of an In Vivo Continuous Ketone Monitoring System

Introduction: Solute binding proteins (SBPs) are promising recognition elements for constructing biosensors due to their selective binding to specific solutes and the conformational changes they undergo upon binding. Focusing on their superb features, we have been working to discover novel SBPs that recognize β-hydroxybutyrate (BHB), designated as BHB binding proteins (BHBBPs). BHB is the major ketone body which is produced during ketosis and diabetic ketoacidosis; therefore, our aim is the development of a continuous BHB sensing system. In order to expand our understanding about the structure/function relationship, particularly regarding the structures/domains/sequences responsible for the specific recognition of BHB, we expanded the investigation of the BHBBP candidate molecule to identify their binding specificities.

Materials and

Methods: Considering homologies with previously discovered BHBBPs (article is in press), multiple BHBBP candidates were recombinantly prepared, and subjected to the following two assays to address their binding specificities: a thermal shift assay to identify relevant compounds that exhibit binding, and an intrinsic tryptophan fluorescence assay to fit binding curves to determine their binding constants.

Results, Conclusions, and Discussions: Our results indicate that most of the candidate BHBBPs exhibited a high degree of specificity for BHB with sub micromolar range of the dissociation constant. Some proteins demonstrated significant binding to other interfering compounds, which may exist in the biological fluid together with BHB. This information will foster our understanding in identifying the specific domain structures of BHBBPs that are responsible for determining the selectivity of their binding and their binding constants. Utilizing this information for protein engineering will enable us to construct an ideal BHBBP with appropriate specificity and sensitivity to be suitable for in vivo continuous BHB monitoring in the physiological range.

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