The loss of cellular proteostasis through aberrant stress granule formation is implicated in neurodegenerative diseases. Stress granules are formed by biomolecular condensation involving protein-protein and protein-RNA interactions. These assemblies are protective, but can rigidify, leading to amyloid-like fibril formation, a hallmark of the disease pathology. Key proteins dictating stress granule formation and disassembly, such as TDP43, contain low-complexity (LC) domains that drive fibril formation. HSPB8, a small heat shock protein, localizes to stress granules, has known aggregation delaying activity, and helps direct aggregated proteins to protein degradation pathways. It is not known how HSPB8 interacts with aggregation prone LC domains in stress granules. Here, we examine the interaction between isolated HSPB8 and the TDP43 LC using thioflavin T (ThT) and fluorescence polarization (FP) aggregation assays, fluorescence microscopy and photobleaching experiments, and crosslinking mass spectrometry (XL-MS). Our results indicate that HSPB8 delays TDP43 LC aggregation through domain-specific interactions with fibril nucleating species, without affecting fibril elongation rates. These findings provide mechanistic insight into how HSPB8 mediates LC domain aggregation and provides bases for investigating how the TDP43 LC subverts chaperone activity in neurodegenerative disease and comparing differing mechanisms between members of the HSPB protein family.

The phosphate-starvation response transcription-factor protein family is essential to plant response to low-levels of phosphate. Proteins in this transcription factor (TF) family act by altering various gene expression levels, such as increasing levels of the acid phosphatase proteins which catalyze the conversion of inorganic phosphates to bio-available compounds. There are few structural characterizations of proteins in this TF family, none of which address the potent TF activation domains. The phosphate-starvation response-like protein-4 (PHL4) protein from this family has garnered interest due to the unusually high TF activation activity of the N-terminal domain. Here, we demonstrate using solution nuclear magnetic resonance (NMR) measurements that the PHL4 N-terminal activating TF effector domain is mainly an intrinsically disordered domain of over 200 residues, and that the C-terminal region of PHL4 is also disordered. Additionally, we present evidence from size-exclusion chromatography, diffusion NMR measurements, and a cross-linking assay suggesting full-length PHL4 forms a trimeric or tetrameric assembly. Together, the data indicate the N- and C-terminal disordered domains in PHL4 flank a central folded region that likely forms the ordered oligomer of PHL4. This work provides a foundation for future studies detailing how the conformations and molecular motions of PHL4 change as it acts as a potent activator of gene expression in phosphate metabolism. Such a detailed mechanistic understanding of TF function will benefit genetic engineering efforts that take advantage of this activity to boost transcriptional activation of genes across different organisms.

The Tropomyosin 1 isoform I/C C-terminal domain (Tm1-LC) fibril structure is studied jointly with cryogenic electron microscopy (cryo-EM) and solid state nuclear magnetic resonance (NMR). This study demonstrates the complementary nature of these two structural biology techniques. Chemical shift assignments from solid state NMR are used to determine the secondary structure at the level of individual amino acids, which is faithfully seen in cryo-EM reconstructions. Additionally, solid state NMR demonstrates that the region not observed in the reconstructed cryo-EM density is primarily in a highly mobile random coil conformation rather than adopting multiple rigid conformations. Overall, this study illustrates the benefit of investigations combining cryo-EM and solid state NMR to investigate protein fibril structure.

Protein domains biased toward a few amino acid types are vital for the formation of biomolecular condensates in living cells. These membraneless compartments are formed by molecules exhibiting a range of molecular motions and structural order. Missense mutations increase condensate persistence lifetimes or structural order, properties that are thought to underlie pathological protein aggregation. In the context of stress granules associated with neurodegenerative diseases, this process involves the rigidification of protein liquid droplets into β-strand rich protein fibrils. Here, we characterize the molecular mechanism underlying the rigidification of liquid droplets for the low complexity domain of the Cytotoxic granule associated RNA binding protein TIA1 (TIA1) stress granule protein and the influence of a disease mutation linked to neurodegenerative diseases. A seeding procedure and solid state nuclear magnetic resonance measurements show that the low complexity domain converges on a β-strand rich fibril conformation composed of 21% of the sequence. Additional solid state nuclear magnetic resonance measurements and difference spectroscopy show that aged liquid droplets of wild type and a proline-to-leucine mutant low complexity domain are composed of fibril assemblies that are conformationally heterogeneous and structurally distinct from the seeded fibril preparation. Regarding low complexity domains, our data support the functional template-driven formation of conformationally homogeneous structures, that rigidification of liquid droplets into conformationally heterogenous structures promotes pathological interactions, and that the effect of disease mutations is more nuanced than increasing thermodynamic stability or increasing β-strand structure content.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Proteins containing low complexity sequence domains harbor mutations genetically linked to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Not much is known about the structures these protein domains adopt functionally and what role the mutations play in causing disease. One key protein with mutations present in the low complexity domain (LCD) is T-cell restricted intracellular antigen-1 (TIA1), which forms fibril-like structures that are usually associated with disease.

Unlocking one third of plant biomass as a renewable feedstock for fuels and materials depends on the effective deconstruction of the secondary plant cell wall. Over 90% of the native secondary plant cell wall is composed of cellulose and hemicellulose polysaccharides and the lignin aromatic polymers. Deconstruction refers to the processes which digest polymers into desired subunits. Present deconstruction processes are centered on lignin-first extraction to overcome the recalcitrance of biomass: the accumulation of indigestible plant polymers during deconstruction.

The condensation of proteins with low complexity sequences plays both functional and pathogenic roles in living cells. Recent efforts to understand the chemistry of biomolecular condensates have revealed that protein sequences harboring weak and dynamic multivalent interaction sites produce the macroscopic behavior observed in living cells. The hydrogen bonding, cation-π, π-π, dipole-dipole, and cation-anion interactions involved are well understood individually, yet fundamental and intriguing questions remain regarding how this chemistry works in specific biological contexts at the structural level.

TDP-43 is an RNA and DNA processing protein with a highly conserved, C-terminal low-complexity domain. In vitro, the TDP-43 low complexity domain can form liquid-liquid phase separations which readily mature into amyloid fibrils. Fibrils containing C-terminal fragments of TDP-43 have been identified within cellular inclusions of post-mortem neuronal tissue in a considerable range of neurodegenerative diseases, including ALS, FTD, and Alzheimer’s. Nearly all ALS-associated mutations are found in the low complexity domain, further highlighting its significance to disease.

Efficient separation of the plant cell wall polymers during lignocellulose processing has been historically challenging due to insolubility of the polymers and their propensity for recalcitrant reassembly. Methods, such as "lignin first" extraction techniques, have advanced efficient biomass use, but the molecular mechanisms for recalcitrance remain enigmatic. Here, we discuss how solid-state Nuclear Magnetic Resonance (NMR) approaches report on the 3D organization of cellulose, xylan, and lignin in the plant cell wall. Recent results illustrate that the organization of these polymers varies across biomass sources and sample preparation methods, with even minimal physical processing causing significant effects. These structural differences contribute to variable extraction efficiencies for bioproducts after downstream processing. We propose that solid-state NMR methods can be applied to follow biomass processing, providing an understanding of the polymer rearrangements that can lead to poor yields for the desired bioproducts. The utility of the technique is illustrated for mechanical processing using lab-scale vibratory ball milling of Sorghum bicolor.

Vimentin is a self-assembling intermediate filament (IF) protein and an integral component of the cell cytoskeleton. Disruptions in the organization and assembly of vimentin IFs have been implicated in over 100 human IF diseases including cataracts, neurodegeneration, and cancer. Despite the known assembly process of vimentin IFs, atomic-level structural characterizations of functionally assembled vimentin IFs are lacking. This project aims to provide high-resolution structural information of intact full-length vimentin IFs by investigating how the tail domain interacts with metals and how these interactions affect the functional higher-order organization of full-length vimentin filaments.

The biomolecular condensation of proteins with low complexity sequences plays a functional role in RNA metabolism and a pathogenic role in neurodegenerative diseases. The formation of dynamic liquid droplets brings biomolecules together to achieve complex cellular functions. The rigidification of liquid droplets into β-strand-rich hydrogel structures composed of protein fibrils is thought to be purely pathological in nature. However, low complexity sequences often harbor multiple fibril-prone regions with delicately balanced functional and pathological interactions. Here, we investigate the maturation of liquid droplets formed by the low complexity domain of the TAR DNA-binding protein 43 (TDP-43). Solid state nuclear magnetic resonance measurements on the aged liquid droplets identify a structured core region distinct from the region thought to be most important for pathological fibril formation and aggregation. The results of this study show that multiple segments of this low complexity domain are prone to form fibrils, and that stabilization of β-strand-rich structure in one segment precludes the other region from adopting rigid fibril structure. Table of Contents Graphic

The biomolecular condensation of proteins with low complexity sequences plays a functional role in RNA metabolism and a pathogenic role in neurodegenerative diseases. The formation of dynamic liquid droplets brings biomolecules together to achieve complex cellular functions. The rigidification of liquid droplets into β-strand-rich hydrogel structures composed of protein fibrils is thought to be purely pathological in nature. However, low complexity sequences often harbor multiple fibril-prone regions with delicately balanced functional and pathological interactions. Here, we investigate the maturation of liquid droplets formed by the low complexity domain of the TAR DNA-binding protein 43 (TDP-43). Solid state nuclear magnetic resonance measurements on the aged liquid droplets identify residues 365-400 as the structured core, which are squarely outside the region between residues 311-360 thought to be most important for pathological fibril formation and aggregation. The results of this study suggest that multiple segments of this low complexity domain are prone to form fibrils and that stabilization of β-strand-rich structure in one segment precludes the other region from adopting a rigid fibril structure.

Sorghum (Sorghum bicolor L. Moench) is a promising source of lignocellulosic biomass for the production of renewable fuels and chemicals, as well as for forage. Understanding secondary cell wall architecture is key to understanding recalcitrance i.e. identifying features which prevent the efficient conversion of complex biomass to simple carbon units. Here, we use multi-dimensional magic angle spinning solid-state NMR to characterize the sorghum secondary cell wall. We show that xylan is mainly in a three-fold screw conformation due to dense arabinosyl substitutions, with close proximity to cellulose. We also show that sorghum secondary cell walls present a high ratio of amorphous to crystalline cellulose as compared to dicots. We propose a model of sorghum cell wall architecture which is dominated by interactions between three-fold screw xylan and amorphous cellulose. This work will aid the design of low-recalcitrance biomass crops, a requirement for a sustainable bioeconomy.