Structural and Biophysical Properties of Low Complexity Proteins

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Two new projects were started in FY2023, with the following preliminary results: (1) DEVELOPMENT OF AN AAO-BASED MIMETIC SYSTEM FOR 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. Preliminary studies with phosphonate-tagged peptides (amyloid-beta, HP35, and a 30-residue peptide from the nuclear pore protein nup98) show that good-quality NMR data can be obtained, that high coverages can be achieved, and that phosphonate-AAO attachments are stable for long time periods at biologically relevant pH values. (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 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 initiated experiments on phase-separated FUS-LC that use the rapid inverse temperature jump technique and low-temperature solid state NMR methods developed in our lab. In preliminary experiments that compare solutions that are rapidly frozen from a monomeric state with solutions that are rapidly frozen from a phase-separated state, we find small but statistically significant differences in 13C solid state NMR signals from certain residues of FUS-LC. These differences may indicate small changes in conformational distributions. We have also observed large differences in nuclear spin relaxation times (specifically, build-up times for 1H spin polarization) that allow us to differentiate the monomeric and phase-separated states, independent of conformational changes. These preliminary results lay the groundwork for planned studies of the kinetics and mechanisms of the phase separation process.