Biology subjects

Murray, D. T.

Publications and source records attributed to Murray, D. T..

11 recordsLinked to original sources

bioRxiv2026

Solid state NMR characterization of wild-type and mutant GFAP intermediate filament assemblies

GFAP is a type III intermediate filament primarily found within astrocytes and is known to maintain proper cell structure and mechanical strength. Mutations in GFAP are implicated in the pathology of Alexander disease, a neurodegenerative disease characterized by cytoplasmic inclusions of protein, known as Rosenthal fibers. GFAP has a typical type III intermediate filament domain structure, consisting of a highly conserved alpha-helical rod domain bracketed by an intrinsically disordered N-terminal head and C-terminal tail domains. While the general domain organization of monomeric GFAP and the assembly process for higher order quaternary structures are known, we lack an atomic resolution mechanistic understanding of GFAP assembly into mature filaments. Understanding the structure of GFAP filaments and how mutations disrupt this structure will provide vital information into how mutations produce Alexander disease pathology. As a first step towards a mechanistic description, we characterized GFAP wild type tetrameric and filamentous assemblies using solid state NMR and compared the results to those obtained from an assembly-deficient GFAP mutant. For wild-type GFAP, we observe surprisingly uniform rigid alpha helical structure and can spectroscopically resolve highly mobile intrinsically disordered regions in the filament assemblies. Wild type tetramers show increased mobility, likely arising from the head and tail domains. Mutation of the highly conserved cysteine at position 294 to serine results in an inability to form full-length filament assemblies. We show that the rigid regions of the C294S mutant assemblies largely remain structurally consistent with wild type tetrameric assemblies but differ from wild-type filament assemblies. There is an increase in highly mobile regions for the C294S mutant relative to the wild-type. Our results provide a foundation for developing solid state NMR approaches to characterize intermediate filament assembly mechanisms and the interfering effect of disease mutations.

biophysics↗

bioRxiv2026

ALS mutations in the TIA1 RNA granule protein have differing effect on low complexity domain fibril formation

Mutations in the low complexity domains of RNA-binding proteins are associated with neurodegenerative disease pathology. The TIA1 RNA-binding protein harbors seven such mutations linked to a clinical cohort of ALS patients. The altered low complexity domain sequence increases the number of TIA1-rich stress granules in cultured cells, delays their disassembly, and is associated with increased fibril formation. Altered molecular motions and contacts in condensed states like stress granules that result in the formation of amyloid-like fibril states is commonly observed for RNA-binding biomolecular condensates. Here we focus on the influence of the ALS mutations on fibril formation of the TIA1 low complexity domain. Repetitive seeding preparations of the seven TIA1 protein mutants all yield amyloid-like fibrils based on transmission electron microscopy images and increased thioflavin T fluorescence. Analysis of solid state nuclear magnetic resonance spectra recorded on all seven mutant fibrils reveals distinct structural differences in the relative to wild-type fibrils. Our results shed light on how the mutations affect structural conformations accessible to the TIA1 low complexity domain.

biophysics↗

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↗

bioRxiv2025

Metal ion binding of vimentin tail domain fragments

The intermediate filament (IF) protein vimentin is widely distributed in various cell types in the body and is vital for the proper maintenance of the cell cytoskeletal architecture, yet an extensive structural characterization of its head and tail domains remains elusive. Alterations in the assembly and organization of vimentin IFs, including filament network reorganization, have been associated with several diseases including cataracts, myopathies, and metastatic cancer. The C-terminal tail domain of vimentin is of increasing interest as it is essential for regulating the structure and mechanical properties of filament networks through interactions with divalent metal ions, but the molecular basis of these tail domain-metal interactions have not been characterized. In this work, we perform an in-depth analysis of the structural and metal binding properties of fragments of the vimentin tail domain. Mass spectrometry, and UV-Vis and circular dichroism (CD) spectroscopy reveal the direct binding of divalent copper (Cu(II)) to the last 11 residues of the tail domain. Solution nuclear magnetic resonance (NMR) and CD measurements show that in isolation, the complete vimentin tail domain is primarily disordered, and that Cu(II)-binding involves both the last 11 residues and another segment in the middle of the tail domain. Aside from these binding sites, Cu(II) does not induce any significant ordering of the tail domain. These findings further support the tail domain serving as a key metal binding region of vimentin and provide new insights into the important interplay between the tail domain and metals in vimentin IF physiology and pathology. O_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC="FIGDIR/small/648257v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@fe39d0org.highwire.dtl.DTLVardef@85958eorg.highwire.dtl.DTLVardef@1dda6corg.highwire.dtl.DTLVardef@1ef0749_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Small heat shock protein HSPB8 interacts with a pre-fibrillar TDP43 low complexity domain species to delay fibril formation

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 examined 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 differing mechanisms between members of the HSPB protein family.

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↗

bioRxiv2022

Liquid droplet aging and fibril formation of the stress granule 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. 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.

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↗

bioRxiv2020

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase

Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur reduction for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit. These structures show how the subunits bind and how both subunit binding and oxidation state impact SiRFPs conformation. Neutron contrast matching experiments on selectively deuterated heterodimers allow us to define the contribution of each subunit to the solution scattering. SiRHP binding induces a change in the position of SiRFPs flavodoxin-like domain relative to its ferredoxin-NADP+ reductase domain while compacting SiRHPs N-terminus. Reduction of SiRFP leads to a more open structure relative to its oxidized state, re-positioning SiRFPs N-terminal flavodoxin-like domain towards the SiRHP binding position. These structures show, for the first time, how both SiRHP binding to, and reduction of, SiRFP positions SiRFP for electron transfer between the subunits.

biochemistry↗

bioRxiv2020

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 upon either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1 IFs functions by forming labile cross-{beta} interactions. These interactions are seen in polymers made from the tail domain alone as well as assembled IFs formed by the intact Tm1 protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology. One Sentence SummaryA new form of protein folding that interconverts between the structured and unstructured states controls assembly of intermediate filaments.

biochemistry↗