Biology subjects

Snead, W. T.

Publications and source records attributed to Snead, W. T..

4 recordsLinked to original sources

bioRxiv2026

Physics-guided design of intrinsically disordered proteins

Intrinsically disordered protein regions (IDPs) are found across the tree of life and characterized by the lack of a stable 3D fold, encoding function through a vast ensemble of conformations. This plasticity makes rational design of IDPs challenging. Physics-based approaches capturing distinct aspects of sequence composition, charge patterning, and molecular interactions have emerged as powerful predictors of ensemble-derived properties. Here, we present a machine learning framework for proteome-scale de novo IDP design by rationally inverting physics-based models. We first program IDPs to tunably sense and respond to diverse biophysical cues and show that IDP ensembles can directly encode complex signal processing, including threshold detection, bandpass filtering, and Boolean-type multi-input logic. We next engineer multicomponent IDP mixtures with tailored emergent condensate properties, including layering and number of phases, compositional specificity, and RNA-dependent remodeling of structure and composition. Finally, we demonstrate designed IDPs that selectively partition into or deplete from biological condensates in living cells. Together, our framework establishes a flexible and scalable strategy for design of ensemble-derived and collective properties in dynamic biomolecules.

biophysics↗

bioRxiv2025

Immiscible proteins compete for RNA binding to order condensate layers

Biomolecular condensates mediate diverse and essential cellular functions by compartmentalizing biochemical pathways. Many condensates have internal subdomains with distinct compositional identities. A major challenge lies in dissecting the multicomponent logic that relates biomolecular features to emergent condensate organization. Nuclear paraspeckles are paradigmatic examples of multi-domain condensates, comprising core and shell layers with distinct compositions that are scaffolded by the lncRNA NEAT1, which spans both layers. A prevailing model of paraspeckle assembly proposes that core proteins bind directly and specifically to core-associated NEAT1 domains. Combining informatics and biochemistry, we unexpectedly find that the essential core proteins FUS and NONO bind and condense preferentially with shell-associated NEAT1 domains. The shell protein TDP-43 exhibits similar NEAT1 domain preferences on its own but forms surfactant-like shell layers around core protein-driven condensates when both are present. Together, experiments and physics-based simulations suggest that competitive RNA binding and immiscibility between core and shell proteins orders paraspeckle layers. More generally, we propose that sub-condensate organization can spontaneously arise from a balance of collaborative and competitive protein binding to the same domains of a lncRNA. SignificanceThe cellular milieu is spatially organized into compartments called biomolecular condensates that exhibit rich internal organization which shapes their functions. Often comprising multiple proteins and RNAs, a major question concerns how molecular-scale features relate to emergent condensate forms. Here we study nuclear paraspeckles, archetypal multi-domain condensates comprising distinct core and shell layers assembled around a layer-spanning RNA scaffold. We find that different proteins associated with each layer all bind preferentially to the same shell-associated domains of the RNA scaffold. Core and shell proteins are inherently immiscible, establishing a competition that redirects core proteins to a suboptimal, core-associated domain of the scaffold. Our work reveals how a combination of competitive RNA binding and protein immiscibility can spatially organize multicomponent condensate subdomains.

biophysics↗

bioRxiv2021

Membrane surfaces regulate assembly of a ribonucleoprotein condensate

Biomolecular condensates organize biochemistry in time and space, yet little is known about how cells control either the position or scale of these assemblies. In cells, condensates often appear as dispersed, relatively small assemblies that do not grow (coarsen) into a single droplet despite their propensity to coalesce. Here we report that ribonucleoprotein condensates of the Q-rich protein Whi3 interact with the endoplasmic reticulum, prompting us to hypothesize that membrane association controls the position and size of condensates. Reconstitution of Whi3 condensates on supported lipid bilayers reveals that association with a diffusive lipid surface promotes condensation at both physiological ionic strength and protein concentration. Notably, these assemblies rapidly arrest, matching size distributions seen in cells. The timing of the arrest is influenced by the ordering of protein-protein and protein-RNA interactions and controlled by the slow diffusion of complexes induced by the membrane. This slowed diffusion limits both transfer of small protein-RNA complexes between condensates and their coalescence, thus driving coarsening to arrest. Our experiments reveal a tradeoff between locally-enhanced protein concentration at membranes, which favors condensation, and an accompanying reduction in diffusion, which restricts coarsening. Thus, membranes can maintain a population of small condensates in the absence of active mechanisms. Given that many condensates are bound to endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell. One sentence summaryAssembly on a membrane surface positions and scales biomolecular condensates by controlling relative diffusion rates of proteins and nucleic acids.

biophysics↗

bioRxiv2021

Negative regulation of a ribonucleoprotein condensate driven by dilute phase oligomerization

Ribonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms ribonucleoprotein condensates with different RNA molecules, and these condensates are implicated in key processes such as cell-cycle control and generating cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region (IDR) and a tandem RNA recognition module. Here, we demonstrate that a 21-residue stretch within the Q-rich IDR has a weak intrinsic preference for forming alpha-helical conformations. Through mutagenesis, we find that increased alpha helicity enhances oligomerization in the dilute phase. One consequence of enhanced oligomerization is a dilution of Whi3 in the dense phase. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, the formation of dilute phase oligomers, driven by a specific sequence motif and potential synergies with the rest of the IDR, opposes incorporation of the Whi3 protein into the dense phase, thereby altering the dense phase stoichiometry of protein to RNA. Our findings, which stand in contrast to other systems where oligomerization has been shown to enhance the drive for phase separation, point to a novel mechanism that might be operative for influencing compositions of condensates. Our work also points to routes for designing synthetic ribonucleoprotein condensates whereby modulation of protein oligomerization via homotypic interactions can impact dense phase concentrations, stoichiometries, and material properties. SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

biophysics↗