Biology subjects

Krol, S.

Publications and source records attributed to Krol, S..

3 recordsLinked to original sources

bioRxiv2021

Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than fifty years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.

biochemistry↗

bioRxiv2021

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic Gram-positive bacterium belonging to the Actinobacteria phylum, which also includes the pathogen Mycobacterium tuberculosis. In the respiratory chain of these bacteria, complexes III (CIII) and IV (CIV) form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. Electron transfer within the CIII2CIV2 supercomplex is linked to transmembrane proton translocation, which maintains an electrochemical proton gradient that drives ATP synthesis and transport processes. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 [A] resolution. The structure shows a central CIII2 dimer flanked by a CIV on each side. One menaquinone is bound in each of the QN and QP sites in each CIII, near the cytoplasmic and periplasmic sides, respectively. In addition, we identified a menaquinone positioned ~14 [A] from heme bL on the periplasmic side. A di-heme cyt. cc subunit provides an electronic connection between each CIII monomer and the adjacent CIV. In CIII2, the Rieske iron-sulfur (FeS) proteins are positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a novel path that conducts protons into CIV.

biochemistry↗

bioRxiv2019

Reproducibility warning: The curious case of Polyethylene glycol 6000 and spheroid cell culture

In this study we report about the reproducibility of three-dimensional cell culture of floating cell spheroids on PEG6000 treated cell culture dishes. Three-dimensional tumour spheroids or organoids present an interesting test platform for nanoparticulated drug delivery or nanoparticle toxicity. We tested the reproducibility of spheroid formation induced by the PEG coated surface. Interestingly we found that the results were different in a reproducible manner depending on the distributors of PEG6000.\n\nDespite the nearly identical physicochemical properties of PEG6000 (MALDI-MS, NMR, FTIR, Triple SEC) with only minor differences, we observed only for one PEG6000 a highly reproducible formation of spheroids with different cell lines such as HT-29, HeLa, Caco2, and PANC-1. The surface coating with the different PEG6000 was studied by AFM. The surface coating as well as the physicochemical characterization showed only small differences in mass and hydrodynamic radius between the different PEGs. A direct coating of the cells with PEG from two distributors indicate that the spheroid formation in due to direct interaction of the polymer with the cell rather than by interaction of cells with the coated surface.\n\nThe experiments point out that for biological entities, such as cells or tissues, even very small differences such as impurities or batch-to-batch variations in the purchased product can have a very strong impact.

scientific communication and education↗