Creation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration.

Point-of-care sensors, which are low-cost and user-friendly, play a crucial role in precision medicine by providing quick results for individuals. Here, we transform the conventional glucometer into a 4-hydroxytamoxifen therapeutic biosensor in which 4-hydroxytamoxifen modulates the electrical signal generated by glucose oxidation. To encode the 4-hydroxytamoxifen signal within glucose oxidation, we introduce the ligand-binding domain of estrogen receptor-alpha into pyrroloquinoline quinone-dependent glucose dehydrogenase by constructing and screening a comprehensive protein insertion library. In addition to obtaining 4-hydroxytamoxifen regulatable engineered proteins, these results unveil the significance of both secondary and quaternary protein structures in propagation of conformational signals. By constructing an effective bioelectrochemical interface, we detect 4-hydroxytamoxifen in human blood samples as changes in the electrical signal and use this to develop an electrochemical algorithm to decode the 4-hydroxytamoxifen signal from glucose. To meet the miniaturization and signal amplification requirements for point-of-care use, we harness power from glucose oxidation to create a self-powered sensor. We also amplify the 4-hydroxytamoxifen signal using an organic electrochemical transistor, resulting in milliampere-level signals. Our work demonstrates a broad interdisciplinary approach to create a biosensor that capitalizes on recent innovations in protein engineering, electrochemical sensing, and electrical engineering.

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion.

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate the binding of MtrA to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). The EET efficiency was found to be inversely correlated with the proximity of the insertion to the heme prosthetic groups. Mutants with decreased EET efficiencies also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular cytochrome MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET efficiencies. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III) respiration conditions correlated with anode reduction. The peptide insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for the regulation of EET.

Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials.

The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(-)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(-)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials.IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

Faster-than-anticipated Na(+)/Cl(-) diffusion across lipid bilayers in vesicles.

Maintenance of electrochemical potential gradients across lipid membranes is critical for signal transduction and energy generation in biological systems. However, because ions with widely varying membrane permeabilities all contribute to the electrostatic potential, it can be difficult to measure the influence of diffusion of a single ion type across the bilayer. To understand the electrodiffusion of H(+) across lipid bilayers, we used a pH-sensitive fluorophore to monitor the lumenal pH in vesicles after a stepwise change in the bulk pH. In vesicles containing the ion channel gramicidin, the lumenal pH rapidly approached the external pH. In contrast, the lumen of intact vesicles showed a two stage pH response: an initial rapid change occurred over ~1min, followed by a much slower change over ~24h. We provide a quantitative interpretation of these results based on the Goldman-Hodgkin-Katz ion fluxes discharging the electrical capacitance of the bilayer membrane. This interpretation provides an estimate of the permeability of the membranes to Na(+) and Cl(-) ions of ~10(-8)cm/s, which is ~3 orders of magnitude faster than previous reports. We discuss possible mechanisms to account for this considerably higher permeability in vesicle membranes.