Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.

The RNA-binding proteins TDP-43 and FUS are tied as the third leading known genetic cause for amyotrophic lateral sclerosis (ALS), and TDP-43 proteopathies are found in nearly all ALS patients. Both the natural function and contribution to pathology for TDP-43 remain unclear. The intersection of functions between TDP-43 and FUS can focus attention for those natural functions mostly likely to be relevant to disease. Here, we compare the role played by TDP-43 and FUS, maintaining chromatin stability for dividing HEK293T cells. We also determine and compare the interactomes of TDP-43 and FUS, quantitating changes in those before and after DNA damage. Finally, selected interactions with known importance to DNA damage repair were validated by co-immunoprecipitation assays. This study uncovered TDP-43 and FUS binding to several factors important to DNA repair mechanisms that can be replication-dependent, -independent, or both. These results provide further evidence that TDP-43 has an important role in DNA stability and provide new ways that TDP-43 can bind to the machinery that guards DNA integrity in cells.

Chemical modification of proteins has been crucial in engineering protein-based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation-based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well-folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with LC-MS/MS. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein..

All cells use organized lipid compartments to facilitate specific biological functions. Membrane‐bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid‐based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)‐modified phospholipids that form stable covalent linkages with an O6‐methylguanine DNA methyltransferase (SNAP‐tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid‐aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP‐tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid‐based artificial organelles.

Many proteins of high interest to biology and biomedical research have few options that can provide even simple insights into structure, such as whether the protein conformation has changed between two states. Tyrosine is an abundant amino acid that balances hydrophobic and hydrophilic properties such that it can be found at protein surfaces, binding interfaces, and buried in a protein core. The reactive compound 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) can conjugate the phenolic side chain of tyrosine. We hypothesized that individual protein conformations could be distinguished by changes induced to tyrosine reactivity with PTAD. We quantified for a wellfolded protein, bovine serum albumen (BSA), the complex distribution of PTAD labeling using LC-MS/MS. As a model conformational switch in BSA, we chose to label unfolded BSA using urea. We found that changes in PTAD reactivity for many tyrosine residues was easily quantified and several sites were highly sensitive to the changes produced by unfolding the protein. This study suggests that the reaction of PTAD to tyrosine may allow for the measurement of a covalent fingerprint that can discriminate conformational states in a protein.