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

Designing Processive Catalytic Systems. Threading Polymers through a Flexible Macrocycle Ring

The translocation of polymers through pores is widely observed in nature and studying their mechanism may help understand the fundamental features of these processes. We describe here the mechanism of threading of a series of polymers through a flexible macrocyclic ring. Detailed kinetic studies show that the translocation speed is slower than the translocation speed through previously described more rigid macrocycles, most likely as a result of the wrapping of the macrocycle around the polymer chain. Temperature-dependent studies reveal that the threading rate increases on decreasing the temperature, resulting in a negative activation enthalpy of threading. The latter is related to the opening of the cavity of the macrocycle at lower temperatures, which facilitates binding. The translocation process along the polymer chain, on the other hand, is enthalpically unfavorable, which can be ascribed to the release of the tight binding of the macrocycle to the chain upon translocation. The combined kinetic and thermodynamic data are analyzed with our previously proposed consecutive-hopping model of threading. Our findings provide valuable insight into the translocation mechanism of macrocycles on polymers, which is of interest for the development of processive catalysts, i.e., catalysts that thread onto polymers and move along it while performing a catalytic action

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

Dynamic Disorder in Single-Enzyme Experiments: Facts and Artifacts

Using a single-molecule fluorescence approach, the time series of catalytic events of an enzymatic reaction can be monitored, yielding a sequence of fluorescent “on”- and “off”-states. An accurate on/off-assignment is complicated by the intrinsic and extrinsic noise in every single-molecule fluorescence experiment. Using simulated data, the performance of the most widely employed binning and thresholding approach was systematically compared to change point analysis. It is shown that the underlying on- and off-histograms as well as the off-autocorrelation are not necessarily extracted from the “signal’’ buried in noise. The shapes of the on- and off-histograms are affected by artifacts introduced by the analysis procedure and depend on the signal-to-noise ratio and the overall fluorescence intensity. For experimental data where the background intensity is not constant over time we consider change point analysis to be more accurate. When using change point analysis for data of the enzyme α-chymotrypsin, no characteristics of dynamic disorder was found. In light of these results, dynamic disorder might not be a general sign of enzymatic reactions

Highly Selective Reduction of Carbon Dioxide to Methane on Novel Mesoporous Rh Catalysts

Mesoporous metals with high surface area hold promise for a variety of catalytic applications, especially for the reduction of CO₂ to value-added products. This study has used a novel mesoporous rhodium (Rh) nanoparticles, which were recently developed via a simple wet chemical reduction approach (<i>Nat. Commun.</i> <b>2017</b>, <i>8</i>, 15581) as catalyst for CO₂ methanation. Highly efficient performance and selectivity for methane formation are achieved due to their controllable crystallinity, high porosity, high surface energy, and large number of atomic steps distributions. The mesoporous Rh nanoparticles, possessing the largest surface area (69 m² g^–1), exhibit a substantially higher reaction rate (5.28 × 10^–5 mol_CO₂ g_Rh^–1 s^–1) than the nonporous Rh nanoparticles (1.28 × 10^–5 mol_CO₂ g_Rh^–1 s^–1). Our results indicate the extensive use of mesoporous metals in heterogeneous catalysis processes

Uncorrelated Dynamical Processes in Tetranuclear Carboxylate Clusters Studied by Variable-Temperature ¹H NMR Spectroscopy.

Tetranuclear carboxylate clusters with the general structural formula [M₄(L)₂(O₂CR)₄] (M = Cd, Zn; LH₂ = 2,6-bis­(1-(2-hydroxyphenyl)-iminoethyl)­pyridine; R = CH₃, C₆H₅) were studied by variable-temperature (VT) ¹H NMR spectroscopy. The dynamics of these clusters in solution can be described by two uncorrelated dynamical processes. The first dynamical process is the interconversion, both inter- as well as intramolecular, between <i>syn</i>–<i>syn</i> bridging and chelating carboxylate ligands. It is shown that this carboxylate interconversion mechanism is predominantly intramolecular for [Cd₄(L)₂(O₂CCH₃)₄] (<b>1a</b>), whereas for [Zn₄(L)₂(O₂CCH₃)₄] (<b>2a</b>) it is predominantly intermolecular. Two models for the second dynamic process, which involves the diiminepyridine ligand, are described. The first model comprises a nondissociative rotation around an internal axis, which changes the chirality of the cluster. The second model is based on the dissociation of the tetranuclear cluster into two dimeric species, which recombine again. This last model is supported by scrambling experiments between [Zn₄(L)₂(O₂CCH₃)₄] (<b>2a</b>) and [Zn₄(L3)₂(O₂CCH₃)₄] (<b>5</b>) (L3H₂ = 2,6-bis­(1-(2-hydroxyphenyl)-iminoethyl)­4-chloropyridine)

Uncorrelated Dynamical Processes in Tetranuclear Carboxylate Clusters Studied by Variable-Temperature ¹H NMR Spectroscopy.

Tetranuclear carboxylate clusters with the general structural formula [M₄(L)₂(O₂CR)₄] (M = Cd, Zn; LH₂ = 2,6-bis­(1-(2-hydroxyphenyl)-iminoethyl)­pyridine; R = CH₃, C₆H₅) were studied by variable-temperature (VT) ¹H NMR spectroscopy. The dynamics of these clusters in solution can be described by two uncorrelated dynamical processes. The first dynamical process is the interconversion, both inter- as well as intramolecular, between <i>syn</i>–<i>syn</i> bridging and chelating carboxylate ligands. It is shown that this carboxylate interconversion mechanism is predominantly intramolecular for [Cd₄(L)₂(O₂CCH₃)₄] (<b>1a</b>), whereas for [Zn₄(L)₂(O₂CCH₃)₄] (<b>2a</b>) it is predominantly intermolecular. Two models for the second dynamic process, which involves the diiminepyridine ligand, are described. The first model comprises a nondissociative rotation around an internal axis, which changes the chirality of the cluster. The second model is based on the dissociation of the tetranuclear cluster into two dimeric species, which recombine again. This last model is supported by scrambling experiments between [Zn₄(L)₂(O₂CCH₃)₄] (<b>2a</b>) and [Zn₄(L3)₂(O₂CCH₃)₄] (<b>5</b>) (L3H₂ = 2,6-bis­(1-(2-hydroxyphenyl)-iminoethyl)­4-chloropyridine)

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

Dibenzo Crown Ether Layer Formation on Muscovite Mica

Stable layers of crown ethers were grown on muscovite mica using the potassium–crown ether interaction. The multilayers were grown from solution and from the vapor phase and were analyzed with atomic force microscopy (AFM), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and surface X-ray diffraction (SXRD). The results show that the first molecular layer of the three investigated dibenzo crown ethers is more rigid than the second because of the strong interaction of the first molecular layer with the potassium ions on the surface of muscovite mica. SXRD measurements revealed that for all of the investigated dibenzo crown ethers the first molecule lies relatively flat whereas the second lies more upright. The SXRD measurements further revealed that the molecules of the first layer of dibenzo-15-crown-5 are on top of a potassium atom, showing that the binding mechanism of this layer is indeed of the coordination complex form. The AFM and SXRD data are in good agreement, and the combination of these techniques is therefore a powerful way to determine the molecular orientation at surfaces