Enzyme purification and sustained enzyme activity for pharmaceutical biocatalysis by fusion with phase-separating intrinsically disordered protein.

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.

Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants.

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase-separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

Designer membraneless organelles sequester native factors for control of cell behavior.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior.

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or "membraneless organelles" such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Particle-Tracking Microrheology Using Micro-Optical Coherence Tomography.

Clinical manifestations of cystic fibrosis (CF) result from an increase in the viscosity of the mucus secreted by epithelial cells that line the airways. Particle-tracking microrheology (PTM) is a widely accepted means of determining the viscoelastic properties of CF mucus, providing an improved understanding of this disease as well as an avenue to assess the efficacies of pharmacologic therapies aimed at decreasing mucus viscosity. Among its advantages, PTM allows the measurement of small volumes, which was recently utilized for an in situ study of CF mucus formed by airway cell cultures. Typically, particle tracks are obtained from fluorescence microscopy video images, although this limits one's ability to distinguish particles by depth in a heterogeneous environment. Here, by performing PTM with high-resolution micro-optical coherence tomography (µOCT), we were able to characterize the viscoelastic properties of mucus, which enables simultaneous measurement of rheology with mucociliary transport parameters that we previously determined using µOCT. We obtained an accurate characterization of dextran solutions and observed a statistically significant difference in the viscosities of mucus secreted by normal and CF human airway cell cultures. We further characterized the effects of noise and imaging parameters on the sensitivity of µOCT-PTM by performing theoretical and numerical analyses, which show that our system can accurately quantify viscosities over the range that is characteristic of CF mucus. As a sensitive rheometry technique that requires very small fluid quantities, µOCT-PTM could also be generally applied to interrogate the viscosity of biological media such as blood or the vitreous humor of the eye in situ.

Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors.

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.

A functional anatomic defect of the cystic fibrosis airway.

RATIONALE: The mechanisms underlying cystic fibrosis (CF) lung disease pathogenesis are unknown. OBJECTIVES: To establish mechanisms linking anion transport with the functional microanatomy, we evaluated normal and CF piglet trachea as well as adult swine trachea in the presence of selective anion inhibitors. METHODS: We investigated airway functional microanatomy using microoptical coherence tomography, a new imaging modality that concurrently quantifies multiple functional parameters of airway epithelium in a colocalized fashion. MEASUREMENTS AND MAIN RESULTS: Tracheal explants from wild-type swine demonstrated a direct link between periciliary liquid (PCL) hydration and mucociliary transport (MCT) rates, a relationship frequently invoked but never experimentally confirmed. However, in CF airways this relationship was completely disrupted, with greater PCL depths associated with slowest transport rates. This disrupted relationship was recapitulated by selectively inhibiting bicarbonate transport in vitro and ex vivo. CF mucus exhibited increased viscosity in situ due to the absence of bicarbonate transport, explaining defective MCT that occurs even in the presence of adequate PCL hydration. CONCLUSIONS: An inherent defect in CF airway surface liquid contributes to delayed MCT beyond that caused by airway dehydration alone and identifies a fundamental mechanism underlying the pathogenesis of CF lung disease in the absence of antecedent infection or inflammation.

Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: demonstration of diamagnetic chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI).

Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging ("theranostics"). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥7 mol% PEG diffused only ~10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7 mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3mol%-PEG liposomes. However, increasing PEG content to ~12 mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7 mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. FROM THE CLINICAL EDITOR: This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.

Expanding the molecular language of protein liquid-liquid phase separation.

Understanding the relationship between a polypeptide sequence and its phase separation has important implications for analysing cellular function, treating disease and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), schematized as a 'molecular grammar' for LLPS. Here we further probe how sequence modulates phase separation and the material properties of the resulting condensates, targeting sequence features previously overlooked in the literature. We generate sequence variants of a repeat polypeptide with either no charged residues, high net charge, no glycine residues or devoid of aromatic or arginine residues. All but one of 12 variants exhibited LLPS, albeit to different extents, despite substantial differences in composition. Furthermore, we find that all the condensates formed behaved like viscous fluids, despite large differences in their viscosities. Our results support the model of multiple interactions between diverse residue pairs-not just a handful of residues-working in tandem to drive the phase separation and dynamics of condensates.

Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy.

Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.

Controlled and orthogonal partitioning of large particles into biomolecular condensates.

Biomolecular condensates arising from liquid-liquid phase separation contribute to diverse cellular processes, such as gene expression. Partitioning of client molecules into condensates is critical to regulating the composition and function of condensates. Previous studies suggest that client size limits partitioning, with dextrans >5 nm excluded from condensates. Here, we asked whether larger particles, such as macromolecular complexes, can partition into condensates based on particle-condensate interactions. We sought to discover the biophysical principles that govern particle inclusion in or exclusion from condensates using polymer nanoparticles with tailored surface chemistries as models of macromolecular complexes. Particles coated with polyethylene glycol (PEG) did not partition into condensates. We next leveraged the PEGylated particles as an inert platform to which we conjugated specific adhesive moieties. Particles functionalized with biotin partitioned into condensates containing streptavidin, driven by high-affinity biotin-streptavidin binding. Oligonucleotide-decorated particles exhibited varying degrees of partitioning into condensates, depending on condensate composition. Partitioning of oligonucleotide-coated particles was tuned by altering salt concentration, oligonucleotide length, and oligonucleotide surface density. Remarkably, beads with distinct surface chemistries partitioned orthogonally into immiscible condensates. Based on our experiments, we conclude that arbitrarily large particles can controllably partition into biomolecular condensates given sufficiently strong condensate-particle interactions, a conclusion also supported by our coarse-grained molecular dynamics simulations and theory. These findings may provide insights into how various cellular processes are achieved based on partitioning of large clients into biomolecular condensates, as well as offer design principles for the development of drug delivery systems that selectively target disease-related biomolecular condensates.

Surface tension and viscosity of protein condensates quantified by micropipette aspiration.

The material properties of biomolecular condensates have been suggested to play important biological and pathological roles. Despite the rapid increase in the number of biomolecules identified that undergo liquid-liquid phase separation, quantitative studies and direct measurements of the material properties of the resulting condensates have been severely lagging behind. Here, we develop a micropipette-based technique that uniquely, to our knowledge, allows quantifications of both the surface tension and viscosity of biomolecular condensates, independent of labeling and surface-wetting effects. We demonstrate the accuracy and versatility of this technique by measuring condensates of LAF-1 RGG domains and a polymer-based aqueous two-phase system. We further confirm our measurements using established condensate fusion and fluorescence recovery after photobleaching assays. We anticipate the micropipette-based technique will be widely applicable to biomolecular condensates and will resolve several limitations regarding current approaches.

Biomolecular Condensates: Sequence Determinants of Phase Separation, Microstructural Organization, Enzymatic Activity, and Material Properties.

This perspective article highlights recent progress and emerging challenges in understanding the formation and function of membraneless organelles (MLOs). A long-term goal in the MLO field is to identify the sequence-encoded rules that dictate the formation of compositionally controlled biomolecular condensates, which cells utilize to perform a wide variety of functions. The molecular organization of the different components within a condensate can vary significantly, ranging from a homogeneous mixture to core-shell droplet structures. We provide many examples to highlight the richness of the observed behavior and potential research directions for improving our mechanistic understanding. The tunable environment within condensates can, in principle, alter enzymatic activity significantly. We examine recent examples where this was demonstrated, including applications in synthetic biology. An important question about MLOs is the role of liquid-like material properties in biological function. We discuss the need for improved quantitative characterization tools and the development of sequence-structure-dynamics relationships.

SPLIT: Stable Protein Coacervation Using a Light Induced Transition.

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.

Controllable protein phase separation and modular recruitment to form responsive membraneless organelles.

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales.

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales - from tens of nanometers to tens of micrometers - using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample using brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. These findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.