Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield TM relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.

The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (k inac/K i = 35,300 M–1s–1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.

No abstract

Bacterial cell wall biosynthesis is the target of many important antibiotics. Its spatiotemporal organization is closely coordinated with cell division. However, the role of peptidoglycan synthesis within cell division is not fully understood. Even less is known about the impact of antibiotics on the coordination of these two essential processes. Visualizing the essential cell division protein FtsZ and other key proteins in Staphylococcus aureus , we show that antibiotics targeting peptidoglycan synthesis arrest cell division within minutes of treatment. The glycopeptides vancomycin and telavancin completely inhibit septum constriction in all phases of cell division. The beta-lactam oxacillin stops division progress by preventing recruitment of the major peptidoglycan synthase PBP2 to the septum, revealing PBP2 as crucial for septum closure. Our work identifies cell division as key cellular target of these antibiotics and provides evidence that peptidoglycan synthesis is the essential driving force of septum constriction throughout cell division of S. aureus .