Biology subjects

Schmit, J. D.

Publications and source records attributed to Schmit, J. D..

15 recordsLinked to original sources

bioRxiv2026

Linker stretching optimizes bonding entropy in sticker-spacer condensates

Phase separating biopolymers are often described by the sticker-spacer model, which accounts for non-attractive linkers that connect discrete attractive moieties. "Associative polymer" theories describe these systems as two coexisting fluids of stickers and spacers, respectively. In such theories the stickers and spacers are decoupled in the sense that sticker binding does not affect spacer conformations or vice versa. Informed by coarse-grained simulations, we develop a phenomenological mean-field theory to determine the microscopic coupling between the sticker and spacer degrees of freedom. Accounting for linkers connecting adjacent spacers introduces free energy contributions due to conformational entropy of the spacer and the binding combinatorics of the stickers. These contributions have opposite dependencies on spacer length because longer spacers allow a greater volume to search for binding partners but incur a greater entropy cost upon bond formation. The length dependent terms from these contributions cancel leaving a length-independent elastic term arising from the fact that a small amount of spacer stretching lowers the free energy by increasing the number of possible binding partners. The cancellation of spacer length from the theory justifies the decoupling approximation and supports mean-field treatments of heterogeneous spacers.

biophysics↗

bioRxiv2025

Sequence-encoded interactions program internal condensate architecture

Many cellular condensates, such as the nucleolus and stress granules, contain multiple coexisting phases with distinct compositions and material properties. This internal organization is crucial for function, yet how it is established remains unclear. Here, using a programmable DNA system, we reveal how molecular interactions can precisely encode multiphase architecture. We find that phase separation drives macromolecules into a semi-dilute regime where subtle differences in homotypic interaction energies are amplified into dominant organizational forces. A critical interaction energy threshold must be overcome to trigger internal demixing, after which molecular partitioning scales near-linearly with interaction strength. This universal relationship is captured by an associative polymer model, and enables engineering of condensates with up to four coexisting phases exhibiting 100-fold differences in viscosity within the same droplet. These design principles extend to RNA-peptide systems, establishing a general framework for how sequence can program hierarchical self-assembly and organize biological matter.

biophysics↗

bioRxiv2025

Biomolecular phase boundaries are described by a solubility product that accounts for variable stoichiometry and soluble oligomers

The solubility product is a rigorous description of the phase boundary for salt precipitation and has previously been shown to qualitatively describe the condensation of biomolecules. Here we present a derivation of the solubility product showing that the solubility product is also a robust description of biomolecule phase boundaries if care is taken to account for soluble oligomers and variable composition within the dense phase. Our calculation describes equilibrium between unbound monomers, the dense phase, and an ensemble of oligomer complexes with significant finite-size contributions to their free energy. The biomolecule phase boundary very nearly resembles the power law predicted by the solubility product when plotted as a function of the monomer concentrations. However, this simple form is concealed by the presence of oligomers in the dilute phase. Accounting for the oligomer ensemble introduces complexities to the power law phase boundary including re-entrant behavior and large shifts for stoichiometrically matched molecules. We show that allowing variable stoichiometry in the dense phase expands the two phase region, which appears as curvature of the phase boundary on a double-logarithmic plot. Furthermore, this curvature can be used to predict variations in the dense phase composition at different points along the phase boundary. Finally, we show how the solubility product power law can be identified in experiments by using dilute phase dissociation constants to account for the oligomer ensemble.

biophysics↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2025

Optically driven control of mechanochemistry and fusion dynamics of biomolecular condensates via thymine dimerization

Phase-separated biomolecular condensates serve as functional elements of biological cells, contribute to protocell formation in prebiotic systems during early life, and represent a distinct class of soft matter with a broad range of potential applications. Understanding and controlling condensate mechanochemistry is critical for their function and material properties. Photochemical processes, such as UV-induced chemical modifications, are ubiquitous in nature and can have both detrimental and constructive impacts on living systems, and are also readily implemented in engineering applications. However, how phase-separated condensate formation influences photochemical processes, and conversely, how photochemical reactions impact condensate dynamics, remains an open question. Combining scanning probe microscopy with optical imaging and control, we developed assays that enable the study of mechanical transitions and fusion dynamics in condensate droplets, revealing that UV-induced thymine dimerization alters condensate nucleation and coalescence. Depending on the frequency and topological arrangement of thymine dimers, particularly the balance between inter- and intrachain crosslinks, UV can induce a transition from liquid-like to solid-like behaviours or lead to aggregate formation. UV treatment also leads to compartmentalization in condensate systems by e.g., promoting the formation of arrested fusion droplets, which are stable against environmental changes. UV illumination can thus be leveraged to program the architecture and material properties of DNA-based biomolecular condensates, with implications for prebiotic chemistry, and bio-inspired engineering.

biophysics↗

bioRxiv2025

Nanometer condensate organization in live cells derived from partitioning measurements

Biomolecules self-organize into membrane-less organelles known as condensates that compartmentalize essential biochemical processes, such as ribosome biogenesis in the nucleolus1-3. Molecular dynamics within condensates are governed by chemical preferences and interaction networks that can imbue nanoscale structure4-7. Such organization is typically inferred from ensemble-averaged measurements, such as scattering and electron microscopy, which reveal molecular arrangements8-14. However, the complexity of cells obscures the interpretability of these techniques, limiting insight into condensate internal structure and roles in macromolecular assembly and transport. Here, we develop an approach to quantify the average microenvironment surrounding specific proteins within condensates in live cells, using thermodynamic principles to interpret the partitioning of designed protein probes. Using this approach, we find that condensates in cells, including the nucleolus, stress granule, and nuclear pore, exhibit spatial inhomogeneity, aligning with emerging views of condensates as networked fluids5,6,15-18. Within the nucleolus, we link spatial inhomogeneity to ribosome biogenesis, which progressively loosens the average local meshwork, facilitating transport of assembled ribosomal subunits. Within the nuclear pore, we find that transporters experience a weaker local meshwork than nucleoporins, consistent with the selective phase model19,20. Together, our approach uncovers a distinct mode of biomolecular control arising from nanoscale structure, which we term microenvironment coupling, whereby internal interaction landscapes shape transport to enable regulation and proofreading.

biophysics↗

bioRxiv2023

Decoding optimal ligand design for multicomponent condensates

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to the UBQLN2-binding surface of Ub or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. Importantly, the extent to which polyubiquitin hubs can promote UBQLN2 phase separation are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates. Highlights[bullet] There is an optimal polyUb ligand architecture/design that promotes multicomponent phase separation, as polyUb hubs whose Ub units are too close together or too far apart are not effective drivers of phase separation for either UBQLN2 450-624 or full-length UBQLN2. [bullet]Theoretical modeling reveals that Ub incurs a significant inclusion energetic penalty that is balanced by polyUbs ability to act as a hub to amplify UBQLN2-UBQLN2 interactions that facilitate phase separation. [bullet]Naturally-occurring M1-linked polyUb chains are optimized to maximize phase separation with UBQLN2. [bullet]Different linkages used in the Ub code deliver biochemical information via Ub-Ub spacing, whereby different outcomes are regulated by the emergent properties of Ub-containing biomolecular condensates. SignificanceBiomolecular condensates are essential for cellular processes and are linked to human diseases when dysregulated. These condensates likely assemble via phase transitions of a few key driver macromolecules and are further modulated by the interactions with ligands. Previous work showed that ligands with one binding site inhibit driver phase transitions whereas ligand hubs comprising several identical binding sites to drivers promote phase transitions. Here, using a library of designed ligand hubs with decreasing or increasing spacings between binding sites and altered binding affinities with drivers, we employ theory and experiments to establish a set of rules that govern how ligand hubs affect driver phase transitions. Our findings reveal that effects of macromolecules can be manipulated through emergent properties of condensates.

biophysics↗

bioRxiv2023

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

Phase separation is a ubiquitous process that compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of complexes below the saturation concentration, the contribution of condensates vs complexes to function is not always clear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3), which pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to the substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX, but enhance the poly-ubiquitination activity of SPOP towards DAXX. This unexpectedly enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of clusters versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate its activity, and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of these cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

biophysics↗

bioRxiv2022

Physical limits to acceleration of chemical reactions inside phase-separated compartments

We present a theoretical analysis of phase separated compartments as a means to facilitate chemical reactions. We find that the attractive interactions that concentrate reactants within the dense phase inhibit reactions by lowering the chemical potential and mobility of the reactants. Therefore, condensed phases are only beneficial if mobility in the condensed phase can be maintained. This can be achieved in multi-step reactions, where the proximity between enzymatic steps results in higher efficiency with less unreacted substrate, but does not increase the reaction rate. Alternatively, mobility can be maintained if recruitment to the condensed phase is driven by multiple attractive moieties that can bind and release independently. However, the spacers necessary to ensure independence between stickers are prone to entangle with the dense phase scaffold. We find an optimal sticker affinity that balances the need for rapid binding/unbinding kinetics and minimal entanglement. Reaction rates can be accelerated by shrinking the size of the dense phase with a corresponding increase in the number of stickers to enhance recruitment.

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↗

bioRxiv2022

Multiphase condensates from a kinetically arrested phase transition

The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

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↗

bioRxiv2020

Structure-function properties in disordered condensates

Biomolecular condensates appear throughout the cell serving a wide variety of functions. Many condensates appear to form by the assembly of multivalent molecules, which produce phase separated networks with liquid-like properties. These networks then recruit client molecules, with the total composition providing functionality. Here we use a model system of poly-SUMO and poly-SIM proteins to understand client-network interactions and find that the structure of the network plays a strong role in defining client recruitment, and thus functionality. The basic unit of assembly in this system is a zipper-like filament composed of alternating poly-SUMO and poly-SIM molecules. These filaments have defects of unsatisfied bonds that allow for both the formation of a 3D network and the recruitment of clients. The filamentous structure constrains the scaffold stoichiometries and the distribution of client recruitment sites that the network can accommodate. This results in a non-monotonic client binding response that can be tuned independently by the client valence and binding energy. These results show how the interactions within liquid states can be disordered yet still contain structural features that provide functionality to the condensate.

biophysics↗

bioRxiv2020

Thermodynamics of Homopeptide Aggregation

Amyloid aggregates are found in many neurodegenerative diseases including Huntingtons, Alzheimers, and prion diseases. The precise role of the aggregates in disease progression has been difficult to elucidate due to the diversity of aggregated states they can adopt. Here we study the formation of fibrils and oligomers by exon 1 of huntingtin protein. We show that the oligomer states are consistent with polymer micelles that are limited in size by the stretching entropy of the polyglutamine region. The model shows how the sequences flanking the amyloid core modulate aggregation behavior. The N17 region promotes aggregation through weakly attractive interactions, while the C38 tail opposes aggregation via steric repulsion. We also show that the energetics of cross-{beta} stacking by polyglutamine would produce fibrils with many alignment defects, but minor perturbations from the flanking sequences are sufficient to reduce the defects to the level observed in experiment. We conclude with a discussion of the implications of this model for other amyloid forming molecules.

biophysics↗

bioRxiv2019

Protein network structure enables switching between liquid and gel states

Biomolecular condensates are emerging as an important organizational principle within living cells. These condensed states are formed by phase separation, yet little is known about how material properties are encoded within the constituent molecules and how the specificity for being in different phases is established. Here we use analytic theory to explain the phase behavior of the cancer-related protein SPOP and its substrate DAXX. Binary mixtures of these molecules have a phase diagram that contains dilute liquid, dense liquid, and gel states. We show that these discrete phases appear due to a competition between SPOP-DAXX and DAXX-DAXX interactions. The stronger SPOP-DAXX interactions dominate at sub-stoichiometric DAXX concentrations leading to the formation of crosslinked gels. The theory shows that the driving force for gel formation is not the binding energy, but rather the entropy of distributing DAXX molecules on the binding sites. At high DAXX concentrations the SPOP-DAXX interactions saturate, which leads to the dissolution of the gel and the appearance of a liquid phase driven by weaker DAXX-DAXX interactions. This competition between interactions allows multiple dense phases to form in a narrow region of parameter space. We propose that the molecular architecture of phase-separating proteins governs the internal structure of dense phases, their material properties and their functions. Analytical theory can reveal these properties on the long length and time scales relevant to biomolecular condensates.

biophysics↗