Deciphering Design Principles of Förster Resonance Energy Transfer-Based Protease Substrates: Thermolysin-Like Protease from Geobacillus stearothermophilus as a Test Case

Protease activity is frequently assayed using short peptides that are equipped with a Förster resonance energy transfer (FRET) reporter system. Many frequently used donor–acceptor pairs are excited in the ultraviolet range and suffer from low extinction coefficients and quantum yields, limiting their usefulness in applications where a high sensitivity is required. A large number of alternative chromophores are available that are excited in the visible range, for example, based on xanthene or cyanine core structures. These alternatives are not only larger in size but also more hydrophobic. Here, we show that the hydrophobicity of these chromophores not only affects the solubility of the resulting FRET-labeled peptides but also their kinetic parameters in a model enzymatic reaction. In detail, we have compared two series of 4–8 amino acid long peptides, designed to serve as substrates for the thermolysin-like protease (TLP-ste) from Geobacillus stearothermophilus. These peptides were equipped with a carboxyfluorescein donor and either Cy5 or its sulfonated derivative Alexa Fluor 647 as the acceptor. We show that the turnover rate <i>k</i>_cat is largely unaffected by the choice of the acceptor fluorophore, whereas the <i>K</i>_M value is significantly lower for the Cy5- than for the Alexa Fluor 647-labeled substrates. TLP-ste is a rather nonspecific protease with a large number of hydrophobic amino acids surrounding the catalytic site, so that the fluorophore itself may form additional interactions with the enzyme. This hypothesis is supported by the result that the difference between Cy5- and Alexa Fluor 647-labeled substrates becomes less pronounced with increasing peptide length, that is, when the fluorophore is positioned at a larger distance from the catalytic site. These results suggest that fluorophores may become an integral part of FRET-labeled peptide substrates and that <i>K</i>_M and <i>k</i>_cat values are generally only valid for a specific combination of the peptide sequence and FRET pair

Induced Mineralization of Hydroxyapatite in Escherichia coli Biofilms and the Potential Role of Bacterial Alkaline Phosphatase

Biofilms appear when bacteria colonize a surface and synthesize and assemble extracellular matrix components. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphate. While calcified biofilms induce diseases like periodontitis in physiological environments, they also inspire the engineering of living composites. Understanding mineralization mechanisms in biofilms will thus provide key knowledge for either inhibiting or promoting mineralization in these research fields. In this work, we study the mineralization of Escherichia coli biofilms using the strain E. coli K-12 W3110, known to produce an amyloid-based fibrous matrix. We first identify the mineralization conditions of biofilms grown on nutritive agar substrates supplemented with calcium ions and β-glycerophosphate. We then localize the mineral phase at different scales using light and scanning electron microscopy in wet conditions as well as X-ray microtomography. Wide-angle X-ray scattering enables us to further identify the mineral as being hydroxyapatite. Considering the major role played by the enzyme alkaline phosphatase (ALP) in calcium phosphate precipitation in mammalian bone tissue, we further test if periplasmic ALP expressed from the phoA gene in E. coli is involved in biofilm mineralization. We show that E. coli biofilms grown on mineralizing medium supplemented with an ALP inhibitor undergo less and delayed mineralization and that purified ALP deposited on mineralizing medium is sufficient to induce mineralization. These results suggest that also bacterial ALP, expressed in E. coli biofilms, can promote mineralization. Overall, knowledge about hydroxyapatite mineralization in E. coli biofilms will benefit the development of strategies against diseases involving calcified biofilms as well as the engineering of biofilm-based living composites

Induced Mineralization of Hydroxyapatite in Escherichia coli Biofilms and the Potential Role of Bacterial Alkaline Phosphatase

Biofilms appear when bacteria colonize a surface and synthesize and assemble extracellular matrix components. In addition to the organic matrix, some biofilms precipitate mineral particles such as calcium phosphate. While calcified biofilms induce diseases like periodontitis in physiological environments, they also inspire the engineering of living composites. Understanding mineralization mechanisms in biofilms will thus provide key knowledge for either inhibiting or promoting mineralization in these research fields. In this work, we study the mineralization of Escherichia coli biofilms using the strain E. coli K-12 W3110, known to produce an amyloid-based fibrous matrix. We first identify the mineralization conditions of biofilms grown on nutritive agar substrates supplemented with calcium ions and β-glycerophosphate. We then localize the mineral phase at different scales using light and scanning electron microscopy in wet conditions as well as X-ray microtomography. Wide-angle X-ray scattering enables us to further identify the mineral as being hydroxyapatite. Considering the major role played by the enzyme alkaline phosphatase (ALP) in calcium phosphate precipitation in mammalian bone tissue, we further test if periplasmic ALP expressed from the phoA gene in E. coli is involved in biofilm mineralization. We show that E. coli biofilms grown on mineralizing medium supplemented with an ALP inhibitor undergo less and delayed mineralization and that purified ALP deposited on mineralizing medium is sufficient to induce mineralization. These results suggest that also bacterial ALP, expressed in E. coli biofilms, can promote mineralization. Overall, knowledge about hydroxyapatite mineralization in E. coli biofilms will benefit the development of strategies against diseases involving calcified biofilms as well as the engineering of biofilm-based living composites

Interfacial Activation of <i>Candida antarctica</i> Lipase B: Combined Evidence from Experiment and Simulation

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. <i>Candida antarctica</i> lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small “lid” (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents

Controlling T‑Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect

Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment

Electrical Monitoring of sp³ Defect Formation in Individual Carbon Nanotubes

Many carbon nanotube (CNT) applications require precisely controlled chemical functionalization that is minimally disruptive to electrical performance. A promising approach is the generation of sp³ hybridized carbon atoms in the sp²-bonded lattice. We have investigated the possibility of using a carboxylic acid-functionalized diazonium reagent to introduce a defined number of sp³ defects into electrically contacted CNTs. Having performed real-time measurements on individually contacted CNTs, we show that the formation of an individual defect is accompanied by an upward jump in resistance of approximately 6 kΩ. Additionally, we observe downward jumps in resistance of the same size, indicating that some defects are unstable. Our results are explained by a two-step reaction mechanism. Isolated aryl groups, formed in the first step, are unstable and dissociate on the minute time scale. Stable defect generation requires a second step: the coupling of a second aryl group adjacent to the first. Additional mechanistic understanding is provided by a systematic investigation of the gate voltage dependence of the reaction, showing that defect formation can be turned on and off. In summary, we demonstrate an unprecedented level of control over sp³ defect formation in electrically contacted CNTs, and prove that sp³ defects are minimally disruptive to the electrical performance of CNTs