Biology subjects

Obermeyer, A. C.

Publications and source records attributed to Obermeyer, A. C..

12 recordsLinked to original sources

bioRxiv2026

Catalytic Bacterial Nanocellulose Composite That Captures and Degrades PET Microplastics

Microplastic (MP) pollution is a growing environmental concern, with wastewater treatment plants (WWTPs) serving as a critical yet insufficient barrier. Although WWTPs can remove up to 99% of MPs from influent streams, a large fraction of the sequestered MPs is concentrated into sewage sludge and subsequently redistributed to soil and waterways via biosolids. Here, we report a biosynthetic approach to address MP redistribution by coupling microplastic removal with catalytic depolymerization, using enzyme-functionalized bacterial nanocellulose (BNC) filtration materials. As a proof-of-concept, we fused a cellulose-binding domain (CBD) to the thermostable PET hydrolase HotPETase, creating a bifunctional protein, CBD-PETase, that binds to a BNC scaffold and degrades PET microplastic ({micro}PET) captured within the scaffold. We showed that BNC-CBD-PETase composites sequestered irregularly shaped {micro}PET within hierarchical pore networks and generated PET degradation products proportionally to enzyme loading. We further established a co-culture strategy, in which Saccharomyces cerevisiae was engineered to secrete CBD-PETase during BNC scaffold biosynthesis by Komagataeibacter xylinus, which enabled one-pot fabrication of an active BNC-CBD-PETase composite without separate protein purification. Notably, co-culture-derived BNC-CBD-PETase composites retained catalytic activity for at least 24 weeks under dry, ambient storage. This platform provides a route towards a scalable and renewable filtration material with the potential for integration across multiple treatment stages within WWTPs.

synthetic biology↗

bioRxiv2026

Dynamics of synthetic transcriptional condensates emerge from RNA synthesis and degradation

At sites of active gene expression, dynamic compartments known as transcriptional condensates assemble and dissolve on timescales relevant to RNA synthesis and degradation. Yet how the non-equilibrium dynamics of these condensates emerge from the coupling of RNA concentration and phase separation remains poorly understood. Here we engineer synthetic active condensates in which T7 RNA polymerase transcribes RNA in situ, triggering phase separation with a cationic scaffold protein. By using RNA concentration as a tunable parameter, we drive condensates along defined paths through a characterized phase diagram. This reaction-phase separation coupling gives rise to three emergent dynamic phenomena not accessible in passive systems: a rapid switch-like nucleation burst, RNA-mediated positive and negative feedback regulation of transcription, and oscillatory condensate formation in which RNA degradation spontaneously renucleates condensates. Together, these results show that the dynamic functions of transcriptional condensates emerge from their reaction-driven paths through phase space, providing a quantitative framework for understanding how RNA flux governs condensate dynamics in living cells.

biophysics↗

bioRxiv2026

Coacervate droplet sequestration of heterogenous nanoplastics with elastin-like polypeptides

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

bioengineering↗

bioRxiv2026

Electrostatic Self-Assembly of Protein Nanoparticles from Intrinsically Disordered Polypeptide and Globular Protein Pairs

Intracellular delivery of folded, functional proteins remains a critical barrier to realizing the full potential of protein therapeutics. We present a fully aqueous, genetically encoded platform for assembling micellar protein nanoparticles via electrostatic coacervation between anionic globular proteins and multi-domain intrinsically disordered proteins (IDPs) composed of a neutral elastin-like polypeptide (ELP) domain and a cationic disordered histone-derived domain (H5). This biosynthetic system allows modular control over charge density, neutral domain length, and stoichiometry, enabling the formation of homogeneous micellar nanoparticles with high protein payload retention under mild, biologically relevant conditions without organic solvents or covalent modification. Unlike amphiphilic micelle systems, globular proteins in this approach act as both cargo and assembly drivers, providing a direct handle to tune encapsulation. Nanoparticle assembly was characterized with a suite of complementary techniques including DLS, FCS, TEM, and SAXS, enabling detailed analysis of nanoparticle structure, size, and composition. The system exhibits remarkable tolerance to different formulations, consistently producing well-defined particles with low dispersity. This expanded design space arises from the use of structured, charge-regulating proteins and confers exceptional versatility in formulation. Different charge fractions modulate not only nanoparticle size but also physicochemical properties such as core density and cargo loading, which may be critical for tuning nanoparticle stability and performance. Together, these features establish a robust and programmable platform for protein-based nanoparticle engineering with potential application in intracellular protein delivery.

bioengineering↗

bioRxiv2025

Cationic amino acid identity and net charge influence condensate properties in E. coli

Understanding the formation of biomolecular condensates (BMC) in biological systems has proven to be a paradigm shift in our understanding of the subcellular organization of biomacromolecules. From RNA metabolism, stress response mechanisms, and amyloidogenic pathologies, condensates have been implicated to play a role in a myriad of cellular phenomena. Despite their near ubiquity, we still do no wholly understand how the primary sequence of biomolecules influences their biophysical and rheological properties. Here, we aim to understand the impact of primary cationic amino acid composition on the properties of condensates. Using engineered recombinant proteins, we show that the formation and phase boundaries of coacervates formed between proteins and RNA is dependent on the cationic amino acid identity, as well as the net charge of the protein involved in condensation. Despite the equivalent charge between arginine and lysine at physiological pH, arginine has been shown to promote increased encapsulation efficiency and salt stability, as well as reduced protein mobility within condensates. We show that arginine-tagged globular proteins also have a higher salt resistance in vitro when compared to similar lysine-tagged globular proteins. This translates to a cellular context in which arginine tagged proteins promote increased condensate formation in model E. coli cells. We were also able to observe a reduction in the total fluorescent recovery and protein mobility within arginine-based condensates via FRAP. Together, these results suggest that in addition to electrostatic interactions and disorder as the main driving forces of phase separation in biological contexts, the primary sequence and side chain composition of proteins plays a significant role in dictating dynamics of coacervates.

biophysics↗

bioRxiv2025

Transient pH changes drive vacuole formation in enzyme-polymer condensates

Intracellular membraneless organelles formed by the phase separation of biomolecules are essential for cellular functioning. These biomolecular condensates often exhibit complex morphologies in response to biological stimuli. In vitro condensate models help elucidate the mechanism of formation and the associated function of these hierarchical assemblies. Here, we use an in vitro model to investigate the formation of hollow internal regions, or vacuoles, within the condensate interior in response to a pH change. Our experimental system is a pH-responsive complex coacervate formed by the anionic glucose oxidase enzyme phase separating with the weak polycation, DEAE-dextran. Fast rates of pH decrease and larger droplet sizes trigger vacuole development within the coacervates. We show that the emergence of vacuoles is a non-equilibrium process caused by the diffusion-limited exchange of condensate components during a fast pH change. We develop a theoretical model that captures how a phase-separating system responds dynamically to changes in system conditions, particularly pH. Our qualitative phase diagram aligns with our experimental results, showing that rapid pH changes shift the phase boundaries, triggering spinodal decomposition and inducing vacuole formation within the condensates. Our pH-sensitive in vitro coacervate model provides a platform to modulate the internal structure of ternary phase separating systems and gain insights into the mechanisms controlling condensate organization in vivo.

biophysics↗

bioRxiv2025

Formation and Gelation of Elastin-like Polypeptide Complex Coacervates

Protein liquid-liquid phase separation underlies the formation of membraneless organelles in cells and performs a key role in the assembly process of natural materials such as the assembly of tropoelastin into elastic fibers. Here, we engineered a series of charged elastin-like polypeptides (ELPs) that form complex coacervates, providing a rapid method to concentrate proteins into a fluid state. Compared to coacervates formed from simple coacervation, complex coacervates exhibited greater fluidity, likely due to differences between electrostatic interactions and hydrophobic forces. We designed these ELPs to further contain crosslinking domains compatible with tyrosinase or transglutaminase and found that crosslinking was enhanced when proteins were in a complex coacervate compared to free in solution. Crosslinking the ELP complex coacervates led to the formation of gels with distinct properties dependent on the nature of the crosslinking. This work expands the design space of ELP hydrogels, offering a novel strategy for forming crosslinked networks from complex coacervates and providing opportunity for future use in tissue engineering and biocompatible biomaterials applications.

synthetic biology↗

bioRxiv2024

Selectivity of Complex Coacervation in Multi-Protein Mixtures

Liquid-liquid phase separation of biomolecules is increasingly recognized as relevant to various cellular functions, and complex coacervation of biomacromolecules, particularly proteins, is emerging as a key mechanism for this phenomenon. Complex coacervation is also being explored as a potential protein purification method due to its potential scalability, aqueous operation, and ability to produce a highly concentrated product. However, to date most studies of complex coacervation have evaluated the phase behavior of a binary mixture of two oppositely charged macromolecules. Therefore, a comprehensive understanding of the phase behavior of complex biological mixtures has yet to be established. To address this, a panel of engineered proteins was designed to allow for quantitative analysis of the complex coacervation of individual proteins within a multi-component mixture. The behavior of individual proteins was evaluated using a defined mixture of proteins that mimics the charge profile of the E. coli proteome. To allow for direct quantification of proteins in each phase, spectrally separated fluorescent proteins were used to construct the protein mixture. From this quantitative analysis, we observed that the coacervation behavior of individual proteins in the mixture was consistent with each other, which was distinctive from the behavior when each protein was evaluated in a single-protein system. Subtle differences in biophysical properties between the proteins became noticeable in the mixture, which allowed us to elucidate parameters for protein complex coacervation. With this understanding, we successfully designed methods to enrich a range of proteins of interest from a mixture of proteins.

biophysics↗

bioRxiv2023

Tuning materials properties of globular protein-based condensates

The phase separation of biomolecules into biomolecular condensates has emerged as a ubiquitous cellular process. Understanding how intrinsically disordered protein sequence controls condensate formation and material properties has provided fundamental biological insights and led to the development of functional synthetic condensates. While these studies provide a valuable framework to understand subcellular organization via phase separation they have largely ignored the presence of folded domains and their impact on condensate properties. We set out to determine how the distribution of sticker interactions across a globular protein contributes to rheological properties of condensates and to what extent globular protein-containing condensates differ from those formed from two disordered components. We designed three variants of green fluorescent protein with different charge patterning and used dynamic light scattering microrheology to measure the viscoelastic spectrum of coacervates formed with poly-lysine over a timescale of 10-6 to 10 seconds, elucidating the response of protein condensates in this range for the first time. We further showed that the phase behavior and rheological characteristics of the condensates varied as a function of both protein charge distribution and polymer/protein ratio, behavior that was distinct to condensates formed with folded domains. Together, this work enhances our fundamental understanding of dynamic condensed biomaterials across biologically relevant length- and time-scales.

biophysics↗

bioRxiv2023

Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria

Subcellular phase separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates in Escherichia coli by utilizing electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates within E. coli cells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria. HighlightsO_LIMinimal disordered cationic peptides of varying charge densities can promote protein phase separation in bacterial cells. C_LIO_LIProtein net charge and charge-patterning are distinct determinants of phase behavior. C_LIO_LIProtein charge distribution can be used to tune the size, number, position, and reversibility of condensates. C_LIO_LIMultiple proteins can be selectively recruited to synthetic condensates with disordered cationic peptides. C_LI

synthetic biology↗

bioRxiv2021

Intracellular phase separation of globular proteins facilitated by short cationic peptides

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We add to this toolbox by designing short, highly charged peptide tags that provide several key advantages for engineering protein phase separation. Herein, we report the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/450573v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1dc5380org.highwire.dtl.DTLVardef@1ee01a9org.highwire.dtl.DTLVardef@af0a85org.highwire.dtl.DTLVardef@1dba73d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Formation of biomolecular condensates in bacteria by tuning protein electrostatics

Biomolecular condensates provide a strategy for cellular organization without a physical membrane barrier while allowing for dynamic, responsive organization of the cell. To date, very few biomolecular condensates have been identified in prokaryotes, presenting an obstacle to engineering these compartments in bacteria. As a novel strategy for bacterial compartmentalization, protein supercharging and complex coacervation were employed to engineer liquid-like condensates in E. coli. A simple model for the phase separation of supercharged proteins was developed and used to predict intracellular condensate formation. Herein, we demonstrate that GFP-dense condensates formed by expressing GFP variants of sufficient charge in cells are dynamic and enrich specific nucleic acid and protein components. This study provides a fundamental characterization of intracellular phase separation in E. coli driven by protein supercharging and highlights future utility in designing functional synthetic membraneless organelles.

synthetic biology↗