Sulfenylation (RSH->RSOH) is a posttranslational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas 'ordinary' trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used (1) H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid (1) H spin-lattice relaxation rate in the rotating frame (R1ρ ), presumably due to intermolecular dipole-dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower (1) H R1ρ . These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.

Protein C‐terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C‐terminal hydrazides in proteins, an efficient intein‐based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C‐terminal hydrazides in protein preparation and modification.

Hydrazides at proteins’ C termini are much desired because of their straightforward use for bioconjugations and chemical modifications. However, traditional preparation methods often lack the necessary biocompatibility. In their full paper, J. Liu, S. Rozovsky, et al. on page 346 in Issue 3, 2020 (DOI: 10.1002/cbic.201900160) show that combining thiols and hydrazine under mild conditions results in the high‐yield generation of such C‐terminal hydrazides. The addition of thiols not only accelerates the formation of the transient thioester intermediate, and the subsequent hydrazinolysis that promotes hydrazide formation at proteins’ C terminus, but also alleviates the problem of destructive protein aggregation. Employing split inteins to trigger hydrazinolysis provides an additional level of control of this now biocompatible generation method.

Viruses, described as ''organisms at the edge of life'', do not have their own metabolism and require a host cell to create multiple copies of themselves. Despite a significant amount of work dedicated to the viral life cycles, there is currently no physical model accounting for the dynamical pathways along which proteins self-assemble around a genome to form the viral capsid. The main goal of this project is to elucidate the dynamical pathways for the assembly and disassembly of hepatitis B virus (HBV) capsids. The wild type HBV capsid protein (HBV Core) is a 183-residue polypeptide comprising an assembly domain (NTD, residues 1-149) and a nucleic acid-binding domain (CTD, residues 150-183). The NTD is necessary and sufficient for self-assembly into icosahedral 120-subunit capsids where the subunit is a dimer of Core. The CTD is necessary and sufficient for interaction with RNA and packaging into the capsid. Using Small angle X-ray scattering (SAXS), we have been probing the assembly and disassembly processes of empty capsids (NTD only) with salinity and chaotropic agent as variables. Using total internal reflection fluorescence microscopy (TIRFM) we study RNA packaging with fluorescently-labelled full-length Core (NTDþCTD) while using fluorescence thermal shift assay we probe the interaction strength between proteins. Taken together, our data allow us to characterize the transition states for self-assembly and disassembly of empty or loaded viral capsids. It would be remarkable to unveil which way HBV has adopted to ensure its survival. Understanding its mechanisms of survival is a key step to spotting its weaknesses in order to promote the development of inhibitors of pregenomic RNA packaging against HBV. 786-PlatAssembly Mechanism of Farnesylated hGBP1 Studied by Time-Resolved Saxs and Electron Microscopy Charlotte Lorenz, Andreas M. Stadler. JCNS-1 & ICS-1, Forschungszentrum J€ ulich, J€ ulich, Germany.Proteins are optimized for interactions with other molecules and proteins, thereby forming the fundamental basis for all kind of functions in organisms. Self-assembly as in aggregation or protein phase-separation is becoming a research focus as malfunctioning can have a severe impact on our lives like in the case of Alzheimer's disease. Our protein of interest is the human Guanylate Binding Protein 1 (hGBP1) that is known for homo-oligomerization and polymerization upon nucleotide activation. After post-translational attachment of a farnesyl lipid as physiologically present in cells, the farnesylated hGBP1 was shown to be involved in immune responses like antimicrobial and antiviral functions by forming 'vesicle-like structures' in vivo that cannot be observed in absence of the farnesyl modification [1]. Using time-resolved ultra-small angle X-ray scattering (TR-USAXS), the polymerization in presence of nucleotides was studied covering a broad size and time range at the beamline ID02 (ESRF). For cross-validation, time-resolved electron microscopy was performed at different stages of the polymerization process....