Discovery of periplasmic solute binding proteins with specificity for ketone bodies: ß-hydroxybutyrate binding proteins.

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 ß-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.

Development of an electrochemical impedance spectroscopy based biosensor for detection of ubiquitin C-Terminal hydrolase L1.

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.

Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide.

INTRODUCTION: The promise of the natural immunoregulator, Galectin-1 (Gal1), as an immunomodulatory therapeutic is challenged by its unstable homodimeric conformation. Previously, a Gal1 homodimer stabilized via covalent poly(ethylene glycol) diacrylate (PEGDA) cross-linking demonstrated higher activity relative to the non-covalent homodimer. METHODS: Here, we report Gal1 homodimers formed using an alternative thiol-Michael addition linker chemistry. RESULTS: Poly(ethylene glycol) bismaleimide (PEGbisMal) reacted with Gal1 at multiple sites with greater efficiency than PEGDA. However, multiple PEGbisMal molecules were conjugated to Gal1 C130, a Gal1 mutant with one surface cysteine (cys-130) and two cysteines thought to be buried in the solvent-inaccessible protein core (cys-42 and cys-60). Site-directed mutagenesis demonstrated that cys-60 was the site at which additional PEGbisMal molecules were conjugated onto Gal1 C130. Compared to WT-Gal1, Gal1 C130 had low activity for inducing Jurkat T cell death, characterized by phosphatidylserine exposure and membrane permeability. PEG cross-linking could restore the function of Gal1 C130, such that at high concentrations Gal1 C130 cross-linked by PEGbisMal had higher activity than both WT-Gal1 and Gal1 C130 cross-linked by PEGDA. Mutating cys-42 and cys-60 to serines in Gal1 C130 did not affect the cell death signaling activity of the Gal1 C130 homodimer cross-linked by PEGbisMal. PEGylated Gal1 C130 variants also eliminated the need for a reducing agent, such as dithiothreitol, which is required to maintain WT-Gal1 signaling activity. CONCLUSION: Collectively, these data demonstrate that thiol-Michael addition bioconjugation leads to a PEG-cross-linked Gal1 homodimer with improved extracellular signaling activity that does not require a reducing environment to be functional.