Biology subjects

Posey, A. E.

Publications and source records attributed to Posey, A. E..

11 recordsLinked to original sources

bioRxiv2024

Biomolecular condensates are characterized by interphase electric potentials

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multi-phase systems defined by coexisting dense and dilute phases. Here, we show that solution ions can partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases of protein and RNA condensates. Asymmetries in ion partitioning between coexisting phases vary with protein sequence, condensate type, salt concentration, and ion type. The Donnan equilibrium set up by asymmetrical partitioning of solution ions generates interphase electric potentials known as Donnan and Nernst potentials. Our measurements show that the interphase potentials of condensates are of the same order of magnitude as membrane potentials of membrane-bound organelles. Interphase potentials quantify the degree to which microenvironments of coexisting phases are different from one another. Importantly, and based on condensate-specific interphase electric potentials, which are membrane-like potentials of membraneless bodies, we reason that condensates are mesoscale capacitors that store charge. Interphase potentials lead to electric double layers at condensate interfaces. This helps explain recent observations of condensate interfaces being electrochemically active.

biophysics↗

bioRxiv2023

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

biophysics↗

bioRxiv2023

Dynamical control enables the formation of demixed biomolecular condensates

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

biophysics↗

bioRxiv2022

The soluble N-termini of mechanosensitive ion channels MSL8, MSL9, and MSL10 are environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations. In the past several decades, IDRs have emerged as a core component of many proteomes, comprising [~]30% of all eukaryotic protein sequences. IDRs are ubiquitous throughout different biological pathways, with a notable enrichment in responses to environmental stimuli such as abiotic stress. However, the diversity of IDR-based systems that biology has evolved to respond to different stimuli is expansive, warranting the exploration of IDRs present in unique molecular contexts. Here, we identify and characterize intrinsic disorder in the soluble, cytoplasmic N-terminal domains of three members of the MscS-Like (MSL) family of mechanosensitive ion channels, MSL8, MSL9 and MSL10. In plants, MSL channels are proposed to mediate the reactions to cell swelling, pathogenic invasion, and touch. A series of bioinformatic tools unanimously predicted that the cytosolic N-termini of MSLs are intrinsically disordered. We confirmed this prediction for the N-terminus of MSL10 (MSL10N) via circular dichroism spectroscopy. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE) and underwent structural changes and alterations to homotypic interaction favorability in the presence of molecular crowding agents. Lastly, in vitro imaging of condensates indicated that MSL8N, MSL9N and MSL10N have sharply differing propensities for condensate formation both inherently and in response to salt, temperature, and molecular crowding. Altogether, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond disparately to changes in their physiochemical environment.

biochemistry↗

bioRxiv2022

Uncovering the contributions of charge regulation to the stability of single alpha helices

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E4K4 repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E4K4 repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.

biophysics↗

bioRxiv2022

Quantifying coexistence concentrations in multi-component phase-separating systems using analytical HPLC

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

biophysics↗

bioRxiv2021

Quantifying charge state heterogeneity for proteins with multiple ionizable residues

Ionizable residues can release and take up protons and this has an influence on protein structure and function. The extent of protonation is linked to the overall pH of the solution and the local environments of ionizable residues. Binding or unbinding of a single proton generates a distinct charge microstate defined by a specific pattern of charges. Accordingly, the overall partition function is a sum over all charge microstates and Boltzmann weights of all conformations associated with each of the charge microstates. This ensemble-of-ensembles description recast as a q-canonical ensemble allows us to analyze and interpret potentiometric titrations that provide information regarding net charge as a function of pH. In the q-canonical ensemble, charge microstates are grouped into mesostates where each mesostate is a collection of microstates of the same net charge. Here, we show that leveraging the structure of the q-canonical ensemble allows us to decouple contributions of net proton binding and release from proton arrangement and conformational considerations. Through application of the q-canonical formalism to analyze potentiometric measurements of net charge in proteins with repetitive patterns of Lys and Glu residues, we are able to determine the underlying mesostate pKa values and, more importantly, we estimate relative mesostate populations as a function of pH. This is a strength of using the q-canonical approach and cannot be obtained using purely site-specific analyses. Overall, our work shows how measurements of charge equilibria, decoupled from measurements of conformational equilibria, and analyzed using the framework of the q-canonical ensemble, provide protein-specific quantitative descriptions of pH-dependent populations of mesostates. This method is of direct relevance for measuring and understanding how different charge states contribute to conformational, binding, and phase equilibria of proteins. STATEMENT OF SIGNIFICANCEThe net charge of a protein in solution is governed by the overall pH as well as context and conformational contexts. Measurements of net charge are accessible via techniques such as potentiometry that quantify the buffering capacity of a protein solution. Here, we use the formal structure of the q-canonical ensemble to identify charge states that are compatible with a measured net charge profile as a function of pH. Our approach highlights how measurements of charge, decoupled from measurements of conformation, can be used to identify the ensembles of charge states that contribute to the overall population for given solution conditions. The methods introduced will be useful for measuring charge states and interpreting these measurements in different contexts.

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↗

bioRxiv2020

Mechanistic inferences from analysis of measurements of protein phase transitions in live cells

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Forster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

biophysics↗

bioRxiv2019

Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation

Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine/aspartic-acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginines guanidinium ion. MCDs can synergise with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behaviour is tuneable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. By contrast, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation, and link the speckles dynamic material properties with function in mRNA processing.

cell biology↗

bioRxiv2018

Evolved sequence features within the intrinsically disordered tail influence FtsZ assembly and bacterial cell division

Intrinsically disordered regions (IDRs) challenge the well-established sequence-structure-function paradigm for describing protein function and evolution. Here, we direct a combination of biophysical and cellular studies to further our understanding of how the intrinsically disordered C-terminal tail of FtsZ contributes to cell division in rod-shaped bacteria. FtsZ is a modular protein that encompasses a conserved GTPase domain and a highly variable intrinsically disordered C-terminal tail (CTT). The CTT is essential for forming the cytokinetic Z-ring. Despite poor sequence conservation of the CTT, the patterning of oppositely charged residues, which refers to the extent of linear mixing / segregation of oppositely charged residues within CTT sequences is bounded within a narrow range. To assess the impact of evolutionary bounds on charge patterning within CTT sequences we performed experiments, aided by sequence design, to quantify the impact of changing the patterning of oppositely charged residues within the CTT on the functions of FtsZ from B. subtilis. Z-ring formation is robust if and only if the extent of linear mixing / segregation of oppositely charged residues within the CTT sequences is within evolutionarily observed bounds. Otherwise, aberrant, CTT-mediated, FtsZ assemblies impair Z-ring formation. The complexities of CTT sequences also have to be above a threshold value because FtsZ variants with low complexity CTTs are not tolerated in cells. Taken together, our results suggest that CTT sequences have evolved to be \"just right\" and that this is achieved through an optimal extent of charge patterning while maintaining the sequence complexity above a threshold value.

biophysics↗