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.

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.

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. We examined seeded fibrils of the T-cell restricted intracellular antigen-1 low complexity domain and determined residues 338-357 compose the rigid fibril core. Aging of wild-type and P362L mutant low complexity domain liquid droplets resulted in fibril assemblies that are structurally distinct from the seeded fibril preparation. The results show that most disease mutations lie outside the region that forms homogeneous fibril structure, the droplets age into conformationally heterogenous fibrils, and the P362L disease mutation does not favor a specific fibril conformation.

Understanding the conformational ensemble of an intrinsically disordered protein (IDP) is of great interest due to its relevance to important intracellular functions and diseases. We have recently shown that the polymer scaling exponent characterizing the dependence of protein size on chain length is a crucial factor as it strongly correlates with liquid-liquid phase behavior of an IDP. Previously, sequence properties from charged amino acids, including both fraction of positive/negative charges and charge patterning have been acknowledged to affect the size of an IDP. However, IDP sequences are composed of a significant amount of uncharged amino acids and how these uncharged amino acids impact the size of an IDP is not well understood. Here, we first investigate if average hydrophobicity can be used to obtain quantitative insights into the polymer scaling properties of IDP sequences. Based on the coarse-grained simulation data for a large number of uncharged IDPs, we find that incorporating the information about the patterning of residues is necessary to model the size of an IDP faithfully. The newly developed sequence hydrophobicity decoration (SHD) parameter, together with the previously known sequence charge decoration (SCD) parameter, can be used to predict the size of an IDP. Our results are, therefore, a significant step forward to elucidate the fundamental principles governing the sequence-structure relationships of disordered proteins.

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