Tuning the Characteristics of β-Hydroxybutyrate Binding Proteins via Site-Directed Mutagenesis

Β-hydroxybutyrate binding proteins (BHBBPs) are a recently discovered group of solute binding proteins (SBPs) derived from bacterial sources that selectively bind to β-hydroxybutyrate, an important biomarker for the detection of diabetic ketoacidosis (DKA). In this research, we build upon recently published work from our group to investigate selected point mutations of these BHBBPs for optimization of binding behavior to expand the library of potential recognition elements for a continuous BHB sensing platform. After analysis of apparent residue coordination with BHB, as assessed in PyMol, we selected a specific residue target, Tyrosine-178 for mutation to serine and alanine, ideally impacting its coordination and binding ability. Once these mutant proteins were recombinantly produced, we assessed specificity of binding with a nano-differential scanning fluorimetry assay, a thermal shift assay allowing for simultaneous testing of 23 potential ligands. The binding affinity of these proteins for BHB was also investigated with a tryptophan fluorescence spectroscopy (TFS) assay, assessing relative inherent tryptophan fluorescence intensity at different ligand concentrations to quantify binding. We found that these point mutations of BHBBPs both reduced binding to differing degrees without appearing to impact binding specificity, expanding the library of biomolecular recognition elements for potential further investigation into a continuous BHB sensing platform.