@article{kane_okuda-shimazaki_andrews_kerrigan_murphy_sode_2024, title={Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β-hydroxybutyrate binding proteins}, volume={33}, ISSN={["1469-896X"]}, url={https://doi.org/10.1002/pro.5025}, DOI={10.1002/pro.5025}, abstractNote={Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as β-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.}, number={7}, journal={PROTEIN SCIENCE}, author={Kane, Bryant J. and Okuda-Shimazaki, Junko and Andrews, Madelyn M. and Kerrigan, Joseph A. and Murphy, Kyle V. and Sode, Koji}, year={2024}, month={Jul} }
 @article{lee_kane_khanwalker_sode_2022, title={Development of an electrochemical impedance spectroscopy based biosensor for detection of ubiquitin C-Terminal hydrolase L1}, volume={208}, ISSN={["1873-4235"]}, url={https://doi.org/10.1016/j.bios.2022.114232}, DOI={10.1016/j.bios.2022.114232}, abstractNote={Year over year, the incidence of traumatic brain injury (TBI) in the population is dramatically increasing; thus, timely diagnosis is crucial for improving patient outcomes in the clinic. Ubiquitin C-terminal hydrolase L1 (UCH-L1), a blood-based biomarker, has been approved by the FDA as a promising quantitative indicator of mild TBI that arises in blood serum shortly after injury. Current gold standard techniques for its quantitation are time-consuming and require specific laboratory equipment. Hence, development of a hand-held device is an attractive alternative. In this study, we report a novel system for rapid, one-step electrochemical UCH-L1 detection. Electrodes were functionalized with anti-UCH-L1 antibody, which was used as a molecular recognition element for selective sensing of UCH-L1. Electrochemical impedance spectroscopy (EIS) was used as a transduction method to quantify its binding. When the electrode was incubated with different concentrations of UCH-L1, impedance signal increased against a concentration gradient with high logarithmic correlation. Upon single-frequency analysis, a second calibration curve with greater signal to noise was obtained, which was used to distinguish physiologically relevant concentrations of UCH-L1. Notably, our system could detect UCH-L1 within 5 min, without a washing step nor bound/free separation, and had resolution across concentrations ranging from 1 pM to 1000 pM within an artificial serum sample. These attributes, together with the miniaturization potential afforded by an impedimetric sensing platform, make this platform an attractive candidate for scale-up as a device for rapid, on-site detection of TBI. These findings may aid in the future development of sensing systems for quantitative TBI detection.}, journal={BIOSENSORS & BIOELECTRONICS}, publisher={Elsevier BV}, author={Lee, Jinhee and Kane, Bryant J. and Khanwalker, Mukund and Sode, Koji}, year={2022}, month={Jul} }