The potent PHL4 transcription factor effector domain contains significant disorder.

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.

Cryo-EM and solid state NMR together provide a more comprehensive structural investigation of protein fibrils.

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.

The Potent PHL4 Transcription Factor Effector Domain Contains Significant Disorder.

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.

Cryo-EM and Solid State NMR Together Provide a More Comprehensive Structural Investigation of Protein Fibrils.

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.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

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.

Liquid Droplet Aging and Seeded Fibril Formation of the Cytotoxic Granule Associated RNA Binding Protein TIA1 Low Complexity Domain.

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.

Solid-State Nuclear Magnetic Resonance as a Tool to Probe the Impact of Mechanical Preprocessing on the Structure and Arrangement of Plant Cell Wall Polymers.

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.

Identification of the Rigid Core for Aged Liquid Droplets of an RNA-Binding Protein Low Complexity Domain.

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.

Transiently structured head domains control intermediate filament assembly.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, β-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order.

A grass-specific cellulose-xylan interaction dominates in sorghum secondary cell walls.

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.

Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments.

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Tm1-I/C IFs functions by forming labile cross-β interactions.

Side Chain Hydrogen-Bonding Interactions within Amyloid-like Fibrils Formed by the Low-Complexity Domain of FUS: Evidence from Solid State Nuclear Magnetic Resonance Spectroscopy.

In aqueous solutions, the 214-residue low-complexity domain of the FUS protein (FUS-LC) is known to undergo liquid-liquid phase separation and also to self-assemble into amyloid-like fibrils. In previous work based on solid state nuclear magnetic resonance (ssNMR) methods, a structural model for the FUS-LC fibril core was developed, showing that residues 39-95 form the fibril core. Unlike fibrils formed by amyloid-β peptides, α-synuclein, and other amyloid-forming proteins, the FUS-LC core is largely devoid of purely hydrophobic amino acid side chains.

Structural characterization of the D290V mutation site in hnRNPA2 low-complexity-domain polymers.

Human genetic studies have given evidence of familial, disease-causing mutations in the analogous amino acid residue shared by three related RNA binding proteins causative of three neurological diseases. Alteration of aspartic acid residue 290 of hnRNPA2 to valine is believed to predispose patients to multisystem proteinopathy. Mutation of aspartic acid 262 of hnRNPA1 to either valine or asparagine has been linked to either amyotrophic lateral sclerosis or multisystem proteinopathy.

Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains.

Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered.

Chaperonin GroEL accelerates protofibril formation and decorates fibrils of the Het-s prion protein.

We have studied the interaction of the prototypical chaperonin GroEL with the prion domain of the Het-s protein using solution and solid-state NMR, electron and atomic force microscopies, and EPR. While GroEL accelerates Het-s protofibril formation by several orders of magnitude, the rate of appearance of fibrils is reduced. GroEL remains bound to Het-s throughout the aggregation process and densely decorates the fibrils at a regular spacing of ∼200 Å.

Membrane protein structural validation by oriented sample solid-state NMR: diacylglycerol kinase.

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers.

Detergent optimized membrane protein reconstitution in liposomes for solid state NMR.

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation.

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples.

Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. Such an environment is often necessary for achieving native-like structures. Sample preparation is the key to this success.

Helical membrane protein conformations and their environment.

Evidence that membrane proteins respond conformationally and functionally to their environment is growing. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment.

Solid state NMR strategy for characterizing native membrane protein structures.

Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment.

Geometry of kinked protein helices from NMR data.

Mathematical questions related to determining the structure of a protein from NMR orientational restraints are discussed. The protein segment is a kinked alpha helix modeled as a regular alpha helix in which two adjacent torsion angles have been varied from their ideal values. Varying these torsion angles breaks the helix into two regular helical segments joined at a kink.