A Dual-Metal-Catalyzed Sequential Cascade Reaction in an Engineered Protein Cage.

Herein, we describe the creation of an artificial protein cage housing a dual-metal-tagged guest protein that catalyzes a linear, two-step sequential cascade reaction. The guest protein consists of a fusion protein of HaloTag and monomeric rhizavidin. Inside the protein capsid, we established a ruthenium-catalyzed allylcarbamate deprotection reaction followed by a gold-catalyzed ring-closing hydroamination reaction that led to indoles and phenanthridines with an overall yield of up to 66 % in aqueous solutions. Furthermore, we show that the encapsulation stabilizes the metal catalysts against deactivation by air, proteins and cell lysate.

Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.

Elastin-like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid-liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least-squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG)20 , (VPGVG)40 , and (VPGVG)60 , we found that coacervation occurs sharply when a certain number of repeat units has acquired ß-turn conformation (in our sequence setting a threshold of approx. 20 repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of ß-turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein-based smart materials.

Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Trehalose Conjugation Enhances Toxicity of Photosensitizers against Mycobacteria.

Trehalose is a natural glucose-derived disaccharide found in the cell wall of mycobacteria. It enters the mycobacterial cell through a highly specific trehalose transporter system. Subsequently, trehalose is equipped with mycolic acid species and is incorporated into the cell wall as trehalose monomycolate or dimycolate. Here, we investigate the phototoxicity of several photosensitizer trehalose conjugates and take advantage of the promiscuity of the extracellular Ag85 complex, which catalyzes the attachment of mycolic acids to trehalose and its analogues. We find that processing by Ag85 enriches and tethers photosensitizer trehalose conjugates directly into the mycomembrane. Irradiation of the conjugates triggers singlet oxygen formation, killing mycobacterial cells more efficiently, as compared to photosensitizers without trehalose conjugation. The conjugates are potent antimycobacterial agents that are, per se, affected neither by permeability issues nor by detoxification mechanisms via drug efflux. They could serve as interesting scaffolds for photodynamic therapy of mycobacterial infections.