Classification of Higher Order Assemblies of FUS and Their Role in Amyotrophic Lateral Sclerosis

Mutations in the nuclear RNA-binding protein Fused in Sarcoma (FUS) gene are responsible for 5% of inherited ALS and 1% of spontaneously acquired ALS. FUS proteins contain an intrinsically disordered low complexity (LC) domain. This domain allows the protein to reversibly self-assemble, undergoing phase transition into condensates. This FUS assembly occurs in an RNA-dependent manner, showing little RNA specificity. These assemblies can then bind the CTD of RNA polymerase II and affect transcription. Mutations within the LC domain of FUS can lead to altered assembly formation. It can also cause protein mislocalization to the cytoplasm. We performed experiments to understand more about both mutagenic and wild type FUS, as there is still much that is unknown. We created mutant primers to establish a library of recombinant FUS plasmids. Each of these primers targeted a tyrosine residue in the LC domain. We also used dynamic light scattering (DLS) to investigate the size and structure of monomeric full length, wild-type FUS. Finally, we performed DLS assays on the FUS to induce assembly formation. These assays allowed for an investigation of both FUS-FUS and FUS-RNA interactions in vitro

Evaluation of the tert-butyl group as a probe for NMR studies of macromolecular complexes

The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.We thank Ad Bax for the suggestion of exploring the use tBu groups as probes for structural studies of the neurotransmitter release machinery, and Ad Bax, Lewis Kay and Charampalos Kalodimos for fruitful discussions on this subject. The Agilent DD2 console of the 800 MHz spectromenter used for the research presented here was purchased with a shared instrumentation grant from the NIH (S10OD018027 to JR). Rashmi Voleti was supported by a fellowship from the Howard Hughes Medical Institute. The preparation of BDSNB was performed at the NANBIOSIS –CIBER BBN Peptide Synthesis Unit (U3). This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).Peer reviewe