Background: The L-cysteine desulfurase IscS interacts with FdhD, a protein essential for the activity of formate dehydrogenases. Results: IscS transfers sulfur to FdhD, and this is required to yield an active enzyme. Conclusion: FdhD is a sulfurtransferase between IscS and formate dehydrogenases. Significance: This helps us to understand how sulfur transfer occurs in living cells.

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Argonaute proteins can be found in all three domains of life. In eukaryotic organisms, Argonaute is, as the functional core of the RNA-silencing machinery, critically involved in the regulation of gene expression. Despite the mechanistic and structural similarities between archaeal, bacterial and eukaryotic Argonaute proteins, the biological function of bacterial and archaeal Argonautes has remained elusive. This review discusses new findings in the field that shed light on the structure and function of Argonaute. We especially focus on archaeal Argonautes when discussing the details of the structural and dynamic features in Argonaute that promote substrate recognition and cleavage, thereby revealing differences and similarities in Argonaute biology.

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post-translationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonaute 2.

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.