Journal: bioRxiv

Article Title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

doi: 10.1101/2021.08.05.455316

Figure Lengend Snippet: (A) Segments of the FUS LC domain containing N-termini at residues 141, 145, 150, 155 and 160, and commonly terminating at residue 214 were purified and incubated under conditions receptive to polymerization (Materials and Methods). (B) Assays for time-dependent acquisition of thioflavin-T fluorescence (left) and electron microscopy (right) were used to monitor the formation of amyloid-like polymers. Scale bar = 200 μM.

Article Snippet: Diluted samples were transferred to a 96 well fluorescence assay plate (Costar assay plate 96, CORNING).

Techniques: Residue, Purification, Incubation, Fluorescence, Electron Microscopy

Journal: bioRxiv

Article Title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

doi: 10.1101/2021.08.05.455316

Figure Lengend Snippet: Acquisition of thioflavin-T fluorescence as a function of time was compared for fragments of the FUS LC domain spanning residues 141-214 bearing either the native sequence or that of variants carrying a single tyrosine-to-serine mutation. Each graph displays fluorescence increase (X-axis) relative to time of incubation (Y axis). Four mutants, including Y143S, Y149S, Y194S and Y208S, revealed evidence of polymerization similar to the wild type protein. Three mutants, including Y155S, Y161S and Y177S, revealed substantially impeded capacity for polymerization.

Article Snippet: Diluted samples were transferred to a 96 well fluorescence assay plate (Costar assay plate 96, CORNING).

Techniques: Fluorescence, Sequencing, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

doi: 10.1101/2021.08.05.455316

Figure Lengend Snippet: (A) Schematic diagram of protein fragments corresponding to N- and C-terminal halves of the FUS low complexity domain. Boxed region shown in yellow corresponds to structural boundaries of the N-terminal cross-β core (NTC) of the FUS LC domain (residues 39-95). Boxed region shown in cyan corresponds to functional boundaries of the C-terminal cross-β core (CTC) forming region of the FUS LC domain (residues 155-180). (B) Proteins fragments shown in panel A were expressed in bacteria, purified and incubated under conditions of neutral pH and physiological monovalent salt for 30 hrs. Thioflavin-T fluorescence (Y axis) was measured as a function of time (X axis). (C) Polymerized samples of NTC and CTC polymers were incubated at varying temperatures (X axis) and monitored for the release of soluble monomers by reverse-phase column chromatography (Y axis).

Article Snippet: Diluted samples were transferred to a 96 well fluorescence assay plate (Costar assay plate 96, CORNING).

Techniques: Functional Assay, Bacteria, Purification, Incubation, Fluorescence, Column Chromatography

Journal: bioRxiv

Article Title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

doi: 10.1101/2021.08.05.455316

Figure Lengend Snippet: (A) Schematic diagram of protein fragments used to express native FUS LC domain (cyan) and ALS-causing variants (G156E = purple; ΔG174/175 = tan). (B) The region spanning residues 141-214 of the FUS LC domain was expressed in its native form as well as when carrying the G156E or ΔG174/G175 ALS-disposing variants. Purified protein was incubated under conditions of neutral pH and physiological monovalent salt for 100 hours. Thioflavin-T fluorescence (Y axis) was measured as a function of time (X-axis), giving evidence of polymerization by the native CTC fragment (blue line), delayed polymerization for the G156E variant (purple line), and no polymerization for the ΔG174/G175 variant (tan line). (C) The region spanning residues 111-214 of the FUS LC domain containing the C-terminal cross-β core was expressed as a GFP fusion in its native form as well as when carrying the G156E or ΔG174/175 ALS-disposing variants. Purified GFP-tagged proteins were incubated with hydrogels formed from mCherry linked to the C-terminal half of the FUS LC domain. Hydrogel binding activity evident for the GFP fusion linked to the native C-terminal half of the FUS LC domain was attenuated for the G156E variant, and absent for the ΔG174/G175 variant.

Article Snippet: Diluted samples were transferred to a 96 well fluorescence assay plate (Costar assay plate 96, CORNING).

Techniques: Purification, Incubation, Fluorescence, Variant Assay, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: The Low Complexity Domain of the FUS RNA Binding Protein Self-assembles via the Mutually Exclusive Use of Two Distinct Cross-β Cores

doi: 10.1101/2021.08.05.455316

Figure Lengend Snippet: (A) Full length derivatives of the FUS low complexity domain bearing the native amino acid sequence (WT), or that carrying either of two ALS-disposing variants, were expressed as 6His-tagged proteins in bacteria, purified and incubated under conditions of neutral pH and physiological monovalent salt in the presence of thioflavin-T. Y axis presents thioflavin-T fluorescence, X-axis presents time of incubation. (B) The same three segments of the FUS low complexity domain were expressed as GFP-tagged proteins in bacteria, purified, and incubated under conditions of neutral pH and physiological monovalent salt with hydrogels formed from mCherry linked to the isolated N-terminal cross-β core of the FUS low complexity domain. Scans depicted to right of hydrogel images present quantitation of GFP signal intensity as measured at hydrogel perimeters. The G156E and ΔG174/G175 variants of the FUS low complexity domain (bottom two rows) displayed between 3- and 4-fold greater binding intensities than that observed for the native FUS protein (top row).

Article Snippet: Diluted samples were transferred to a 96 well fluorescence assay plate (Costar assay plate 96, CORNING).

Techniques: Sequencing, Bacteria, Purification, Incubation, Fluorescence, Isolation, Quantitation Assay, Binding Assay