Biology subjects

Fonda, B. D.

Publications and source records attributed to Fonda, B. D..

4 recordsLinked to original sources

bioRxiv2026

Phosphorylation Mimicking Mutations Cause TDP-43 to Adopt Different Fibril Conformations

The Tar-DNA Binding Protein-43 C-terminal region, TDP43LC, has been previously shown to form amyloid-like fibrils with distinct folds in ALS and FTD. In both diseases, proteinaceous inclusions contain TDP43 C-terminal protein fragments as well as phosphorylated TDP43. Here, we use solution NMR to show that soluble phosphomimetic TDP43LC, P-TDP43LC, is structurally similar to wild-type TDP43LC. Disperse P-TDP43LC, like wild-type protein, contains a central helical region flanked by long disordered regions. Despite this similarity, our turbidity measurements, imaging, and kinetic assays show that P-TDP43LC has different aggregation behavior than wild-type protein. Using solid state NMR measurements we find that that phosphomimetic mutations alter the wild-type fibril conformation. Electrostatic repulsion from negatively charged sidechains, despite having little effect on the soluble proteins structure, perturbs amyloid-like fibril formation and selects for a different conformation in vitro. These results shed light on the structural role of TDP43LC phosphorylation in fibril formation in disease. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/725298v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1e59162org.highwire.dtl.DTLVardef@abe603org.highwire.dtl.DTLVardef@189dfb6org.highwire.dtl.DTLVardef@be6203_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisPhosphomimetic mutations at ALS and FTD neurodegeneration-associated sites in an amyloid forming protein perturbs the aggregated structure compared to wild-type protein.

biophysics↗

bioRxiv2024

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. 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 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. SignificanceTranscription factor proteins upregulate genes and are essential to concerted biological response to environmental conditions like stress or low nutrient availability. In this work, we show the activating effector domain of the potent PHL4 transcription factor protein is primarily disordered, without well-defined secondary structure, and that the isolated effector domain behaves similarly in isolation as it does in the full-length protein. Our finding is consistent with protein transcription factors often having regions of disorder within their functional activator domains.

biophysics↗

bioRxiv2024

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. 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. SignificanceThe use of multiple techniques to structurally characterize proteins provides models that accurately describe molecular conformations better than a technique used in isolation. Combination approaches allow for the study of proteins not only as rigid objects, but rather dynamic molecules that "breathe" over time. Cryogenic electron microscopy and solid state nuclear magnetic resonance are used jointly to provide a more detailed model of the same protein fibrils, and each technique provides novel insights.

biophysics↗

bioRxiv2021

Identification of the Rigid Core for Aged Liquid Droplets of the TDP-43 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 {beta}-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 {beta}-strand-rich structure in one segment precludes the other region from adopting rigid fibril structure. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/433427v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5b0decorg.highwire.dtl.DTLVardef@1ef3887org.highwire.dtl.DTLVardef@684f0aorg.highwire.dtl.DTLVardef@1d23fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗