Biology subjects

Phan, T. M.

Publications and source records attributed to Phan, T. M..

10 recordsLinked to original sources

bioRxiv2026

Scaffold Affinity Tunes Biomolecular Condensate Function

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

biochemistry↗

bioRxiv2026

The role of electrostatic interactions in the phase separation of HP1α and its protein binding partners

Heterochromatin protein 1 (HP1) is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1s binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts. Statement of significanceCellular nuclei are organized into euchromatin and heterochromatin domains, encompassing actively transcribed genes or transcriptionally silent regions, respectively. The regulation of these domains is an active area of study with vast consequences for human health and disease. Here, we focus on the HP1 protein, a key component of heterochromatin compartments that is believed to function through a phase separation mechanism. HP1 interacts with a multitude of binding partners that recognize a specific interaction surface on the protein. While these specific interactions are important, our results suggest that the overall electrostatic network that forms between the proteins has a profound effect on the properties of HP1. These results have important implications for understanding how HP1 function is regulated in heterochromatin environments.

biophysics↗

bioRxiv2025

Multiscale simulations elucidate the mechanism of polyglutamine aggregation and the role of flanking domains in fibril polymorphism

Protein aggregation, which is implicated in aging and neurodegenerative diseases, typically involves a transition from soluble monomers and oligomers to insoluble fibrils. Polyglutamine (polyQ) tracts in proteins can form amyloid fibrils, which are linked to polyQ diseases, including Huntingtons disease (HD), where the length of the polyQ tract inversely correlates with the age of onset. Despite significant research on the mechanisms of Httex1 aggregation, atomistic information regarding the intermediate stages of its fibrillation and the morphological characteristics of the end-state amyloid fibrils remains limited. Recently, molecular dynamics (MD) simulations based on a hybrid multistate structure-based model, Multi-eGO, have shown promise in capturing the kinetics and mechanism of amyloid fibrillation with high computational efficiency while achieving qualitative agreement with experiments. Here, we utilize the multi-eGO simulation methodology to study the mechanism and kinetics of polyQ fibrillation and the effect of the N17 flanking domain of Huntingtin protein. Aggregation simulations of polyQ produced highly heterogeneous amyloid fibrils with variable-width branched morphologies by incorporating combinations of {beta}-turn, {beta}-arc, and {beta}-strand structures, while the presence of the N17 flanking domain reduces amyloid fibril heterogeneity by favoring {beta}-strand conformations. Our simulations reveal that the presence of N17 domain enhanced aggregation kinetics by promoting the formation of large, structurally stable oligomers. Furthermore, the early-stage aggregation process involves two distinct mechanisms: backbone interactions driving {beta}-sheet formation and side-chain interdigitation. Overall, our study provides detailed insights into fibrillation kinetics, mechanisms, and end-state polymorphism associated with Httex1 amyloid aggregation. SIGNIFICANCE STATEMENTPolyglutamine (polyQ) aggregation is central to Huntingtons disease and related neurodegenerative disorders. Despite extensive experimental efforts, a complete molecular understanding of this process--from early aggregation events to the origins of fibril polymorphism--has remained elusive, with varied interpretations of complex fibril architectures. Through multiscale simulations, we reveal how polyQ fibrils adopt diverse tertiary and quaternary structures and demonstrate how the N-terminal flanking domain (N17) modulates fibril architecture and accelerates aggregation. Our hybrid multi-eGO simulations capture early-stage fibrillation kinetics and identify distinct structural polymorphs that align with experimental observations. This work provides a molecular framework for understanding amyloid polymorphism and illuminates the role of flanking domains in shaping aggregation pathways-- offering valuable insights for therapeutic strategies targeting early toxic intermediates.

biophysics↗

bioRxiv2025

Tonotopic Specialization of MYO7A Isoforms in Auditory Hair Cells

1.Mutations in Myo7a cause Usher syndrome type 1B and non-syndromic deafness, but the precise function of MYO7A in sensory hair cells remains unclear. We identify and characterize a novel isoform, MYO7A-N, expressed in auditory hair cells alongside the canonical MYO7A-C. Isoform-specific knock-in mice reveal that inner hair cells primarily express MYO7A-C, while outer hair cells express both isoforms in opposing tonotopic gradients. Both localize to the upper tip-link insertion site, consistent with a role in the tip link for mechanotransduction. Loss of MYO7A-N leads to outer hair cell degeneration and progressive hearing loss. Cryo-EM structures reveal isoform-specific differences at actomyosin interfaces, correlating with distinct ATPase activities. These findings reveal an unexpected layer of molecular diversity within the mechanotransduction machinery. We propose that MYO7A isoform specialization enables fine-tuning of tip-link tension, thus hearing sensitivity, and contributes to the frequency-resolving power of the cochlea.

neuroscience↗

bioRxiv2024

Transient interdomain interactions modulate the monomeric structural ensemble and oligomerization landscape of Huntingtin Exon 1

Polyglutamine expansion ([≥] 36 residues) within the N-terminal exon-1 of Huntingtin (Httex1) leads to Huntingtons disease, a neurodegenerative condition marked by the presence of intranuclear Htt inclusions. Notably, the polyglutamine tract in Httex1 is flanked by an N-terminal coiled-coil domain - N17 (17 amino acids), which undergoes self-association to promote the formation of soluble Httex1 oligomers and brings the aggregation-prone polyQ tracts in close spatial proximity. However, the mechanisms underlying the subsequent conversion of soluble oligomers into insoluble {beta}-rich aggregates with increasing polyQ length, remain unclear. Current knowledge suggests that expansion of the polyQ tract increases its helicity, and this favors its oligomerization and aggregation. In addition, studies utilizing photocrosslinking, conformation-specific antibodies and a stable coiled-coil heterotetrametric system fused to polyQ indicate that domain "cross-talk" (i.e., interdomain interactions) may play a role in the emergence of toxic conformations and the conversion of Httex1 oligomers into fibrillar aggregates. Here, we performed extensive atomistic molecular dynamics (MD) simulations (aggregate time [~] 0.7 ms) to uncover the interplay between structural transformation and domain "cross-talk" on the conformational ensemble and oligomerization landscape of Httex1. Notably, our MD-derived ensembles of N17-polyQ monomers validated against 13C NMR chemical shifts indicated that in addition to elevated - helicity, polyQ expansion also favors transient, interdomain (N17-polyQ) interactions which result in the emergence of {beta}-sheet conformations. Further, interdomain interactions competed with increased polyQ tract -helicity to modulate the stability of N17-mediated dimers and thereby promoted a heterogenous dimerization landscape. Finally, we observed that the C-terminal proline-rich domain (PRD) promoted condensation of Httex1 through self-interactions involving its P10/P11 tracts while also interacting with N17 to suppress its -helicity. In summary, our study demonstrates a significant role for domain "cross-talk" in modulating the monomeric structural ensemble and self-assembly of Httex1.

biophysics↗

bioRxiv2023

How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization

Biomolecular condensates are increasingly appreciated for their function in organizing and regulating biochemical processes in cells, including chromatin function. Condensate formation and properties are encoded in protein sequence but the mechanisms linking sequence to macroscale properties are incompletely understood. Cross species comparisons can reveal mechanisms either because they identify conserved functions or because they point to important differences. Here we use in vitro reconstitution and molecular dynamics simulations to compare Drosophila and human sequences that regulate condensate formation driven by the sterile alpha motif (SAM) oligomerization domain in the Polyhomeotic (Ph) subunit of the chromatin regulatory complex PRC1. We discover evolutionarily diverged contacts between the conserved SAM and the disordered linker that connects it to the rest of Ph. Linker-SAM interactions increase oligomerization and regulate formation and properties of reconstituted condensates. Oligomerization affects condensate dynamics but, in most cases, has little effect on their formation. Linker-SAM interactions also affect condensate formation in Drosophila and human cells, and growth in Drosophila imaginal discs. Our data show how evolutionary sequence changes in linkers connecting conserved structured domains can alter condensate properties. In briefLinking sequence to macroscale properties of biomolecular condensates remains elusive. The authors dissect the function of a disordered linker connecting conserved structured domains in a Polycomb protein with biochemistry and molecular dynamics simulations revealing how sequence changes between Drosophila and humans alter condensates and growth in cells and developing flies. O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/564264v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@a8bd84org.highwire.dtl.DTLVardef@322eaeorg.highwire.dtl.DTLVardef@96a3a8org.highwire.dtl.DTLVardef@1d2a978_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIPRC1 condensates form partly through the conserved SAM domain of Polyhomeotic (Ph) C_LIO_LILinker connecting Ph domains regulates SAM oligomerization and phase separation C_LIO_LILinker-SAM contacts with human but not fly linker in molecular dynamics simulations C_LIO_LILinker-SAM contacts tune condensates in vitro and in cells, and affect cell growth C_LI

biochemistry↗

bioRxiv2023

Interplay between charge distribution and DNA in shaping HP1 paralog phase separation and localization

AbstractThe heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1, HP1{beta}, and HP1{gamma}, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1 with HP1{beta} and HP1{gamma} versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/542535v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@553e82org.highwire.dtl.DTLVardef@7add7dorg.highwire.dtl.DTLVardef@637549org.highwire.dtl.DTLVardef@1a9236a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Molecular interactions underlying the phase separation of HP1: Role of phosphorylation, ligand and nucleic acid binding

Heterochromatin protein 1 (HP1) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1 functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1 can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1 and that it involves interactions between the positively charged hinge region of HP1 and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1 under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1 binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1 can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1 dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1 where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1 and that can contribute to heterochromatin regulation and function.

biophysics↗

bioRxiv2022

Beneficial and Detrimental Effects of Nonspecific Binding During DNA Hybridization

DNA strands have to sample numerous states to find the alignment that maximizes Watson-Crick-Franklin base pairing. This process depends strongly on sequence, which affects the stability of the native duplex as well as the prevalence of non-native inter- and intra-molecular helices. We present a theory which describes DNA hybridization as a three stage process: diffusion, registry search, and zipping. We find that non-specific binding affects each of these stages in different ways. Mis-registered intermolecular binding in the registry search stage helps DNA strands sample different alignments and accelerates the hybridization rate. Non-native intramolecular structure affects all three stages by rendering portions of the molecule inert to intermolecular association, limiting mis-registered alignments to be sampled, and impeding the zipping process. Once inregister base-pairs are formed, the stability of the native structure is important to hold the molecules together long enough for non-native contacts to break.

biophysics↗

bioRxiv2020

Conformational entropy limits the transition from nucleation to elongation in amyloid aggregation

The formation of {beta}-sheet rich amyloid fibrils in Alzheimers disease and other neurodegenerative disorders is limited by a slow nucleation event. To understand the initial formation of {beta}-sheets from disordered peptides, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with {beta} and non-{beta} states. We show that translational and conformational entropy give the nascent {beta}-sheet an anisotropic surface tension which can be used to describe the nucleus with two-dimensional Classical Nucleation Theory. Since translational entropy depends on concentration, the aspect ratio of the critical {beta}-sheet changes with protein concentration. Our model explains the transition from the nucleation phase to elongation as the point where the {beta}-sheet core becomes large enough to overcome the conformational entropy cost to straighten the terminal molecule. At this point the {beta}-strands in the nucleus spontaneously elongate, which results in a larger binding surface to capture new molecules. These results suggest that nucleation is relatively insensitive to sequence differences in co-aggregation experiments because the nucleus only involves a small portion of the peptide. SIGNIFICANCEThe conversion of soluble proteins to amyloid aggregates is associated with many neurodegenerative diseases. Experiments have shown that this conversion occurs by a slow nucleation step followed by rapid growth. This work identifies the principle contributions to the free energy barrier that separates these two stages. It also shows how factors like protein concentration, sidechain interactions, and interactions with the environment can modify the barrier and affect nucleation times.

biophysics↗