Structural and Biophysical Properties of Low Complexity Proteins

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

In FY2025, substantial progress was made on the following two projects: (1) DEVELOPMENT OF AN AAO-BASED MIMETIC SYSTEM FOR FG-REPEAT PROTEINS IN NUCLEAR PORES: Nuclear pores in eukaryotic cells are filled with low complexity protein chains formed by FG-repeat domains of nuclear pore proteins, so called because they contain multiple Phe-Gly motifs. It has been shown previously that FG-repeat sequences are prone to amyloid-like aggregation, leading to hydrogel formation by recombinant FG-repeat constructs in solution. However, the properties of FG-repeat sequences within nuclear pores are not well known. In principle, structural and dynamical properties could be studies by NMR methods, but NMR methods require relatively large quantities (approaching 100 nanomoles) and isotopic labeling. As a potential approach to studying FG-repeat sequences in a pore-like environment, we have developed methods for attaching peptides and proteins to the inner walls of anodic aluminum oxide (AAO) wafers, based on tagging with phosphonate groups through several chemical approaches. The AAO wafers are commercially available inorganic disks with regular arrays of nanopores (20-200 nm diameter, 10-50 micron length) that provide high total surface areas to which NMR-compatible quantities of peptides and proteins can be attached. In FY2024, we optimized conditions for attaching peptides to inner walls of AAO pores in order to maximize the loading within the pores. Based on extensive measurements with various conditions, we are now able to load about 0.1 micromole of a 3 kDa peptide in one AA0 wafer (about 15 mg of AAO). This corresponds to local peptide concentrations of 90 mM or 300 mg/ml within the pores, in other words very high local concentrations, quite comparable to the density of FG-repeat proteins within real nuclear pores. With such high concentrations, we can obtain high-quality 1D and 2D solid state NMR data for 13C-labeled peptides. In FY2025, we carried out an extensive series of spin relaxation measurements to probe temperature-dependent motions of 30-residue peptide that includes four FG motifs, showing that the peptide behaves in a dynamic, random-coil-like manner over a temperature range from -5 C to +50 C, with no signs of aggregation, oligomerization, or phase transition. We found by electron microscopy and solid state NMR that the same peptide readily aggregates in free solution at concentrations above 2 mM, forming amyloid-like fibrils. Thus, tethering to the inner walls of a nanopore dramatically inhibits aggregation, even at extremely high concentrations. We also performed confocal fluorescence microscopy on peptide-loaded AAO wafers, allowing us to determine the average intra-pore concentrations as a function of distance along the pore axis. These results are described in a manuscript that will be submitted before the end of FY2025. (2) LIQUID-LIQUID PHASE SEPARATION BY THE LOW COMPLEXITY DOMAIN OF FUS: The RNA-binding protein FUS has a 214-residue low complexity domain (FUS-LC) which undergoes liquid-liquid phase separation (LLPS) and subsequent fibril formation. Previous studies in our lab by solid state NMR and cryo-EM produced full molecular structures of fibrils formed by full-length FUS-LC and by its C-terminal half. In these structures, certain segments form the in-register parallel beta-sheets of a well-ordered cross-beta motif. As a monomer, FUS-LC is unstructured. The conformational properties of FUS-LC in its phase-separated "droplet" state have not been well characterized, but are believed by others to be essentially the same as in its monomeric state. To investigate this issue, we have performed a series of experiments in which FUS-LC solutions were rapidly frozen from temperatures well above (50 C) or well below (4 C) the LLPS transition temperature (20-30 C, depending on precise conditions). Solid state NMR measurements were then performed on the rapidly frozen samples, which serve as snapshots of the monomeric and phase-separated states, respectively. 2D solid state NMR spectra were recorded for frozen solutions of uniformly 15N,13C-labeled FUS-LC, FUS-LC with 15N,13C-labeling of only Tyr, Thr, and Gly residues, and a selectively labeled construct representing residues 61-214 of FUS, which was produced by native chemical ligation of a synthetic peptide that was labeled only at Gly65, Gln69, Thr78, Tyr81, and Ser86 with an unlabeled segment. In all cases, the 2D spectra of rapidly frozen samples were nearly the same for the monomeric and phase-separated states. Thus, our data show conclusively that local conformational distributions of this low-complexity protein are very similar in the two states, i.e., that phase separation does not involve large changes in secondary structure propensities. Small differences were identified for certain solid state NMR signals, which may indicate a redistribution of populations among different local conformations, less than 20% of the total population. A paper describing these results was published in the Biophysical Journal.