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An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle's size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics.

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multi-component mixtures of biomolecules (e.g. proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g. in inter-molecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g. temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli, or progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of ageing. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically ageing condensates with non-conservative inter-molecular forces. Our nonequilibrium simulations of condensate ageing predict that single-component condensates that are initially homogeneous and liquid-like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age, due to gradual and irreversible enhancement of inter-protein interactions. The type of multiphase architecture is determined by the ageing mechanism, the molecular organization of the gel and liquid phases, and the chemical make up of the protein. Notably, we predict that inter-protein disorder-to-order transitions within the prion-like domains of intracellular proteins could lead to the required non-conservative enhancement of inter-molecular interactions. Our study, therefore, predicts a potential mechanism Significance Statement Biomolecular condensates are highly diverse systems spanning not only homogeneous liquid droplets, but also gels, glasses, and even multiphase architectures that contain various coexisting liquid-like and/or gel-like inner phases. Multiphase architectures form when the different biomolecular components in a multi-component condensate establish sufficiently imbalanced inter-molecular forces to sustain different coexisting phases. While such a requirement seems, at first glance, impossible to fulfil for a condensate formed exclusively of chemically-identical proteins (i.e., single-component), our simulations predict conditions under which this may be possible. During condensate ageing, a sufficiently large imbalance in inter-molecular interactions can emerge intrinsically from the accumulation of protein structural transitions—driving even single-component condensates into nonequilibrium liquid-core/gel-shell or gel-core/liquid-shell multiphase architectures., Adiran Garaizar acknowledges funding from the EPRSC (EP/N509620)

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation., Horizon 2020 programme (766972-FET-OPEN-NANOPHLOW); EPSRC EP/L015889/1; Horizon 2020 Framework Programme through the Marie Sklodowska-Curie grant MicroSPARK (agreement n◦ 841466;, the Herchel Smith Funds, and the Wolfson College Junior Research Fellowship

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Funder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269, Funder: Newman Foundation, Funder: BBSRC; Id: http://dx.doi.org/10.13039/501100000268, Funder: Cambridge Trust; Id: http://dx.doi.org/10.13039/501100003343, Funder: Jardine Foundation, Funder: Trinity College Cambridge, Funder: Trinity College Krishnan‐Ang Studentship, Funder: Honorary Trinity‐Henry Barlow Scholarship, Funder: China Scholarship Council; Id: http://dx.doi.org/10.13039/501100004543, Funder: Herchel Smith Funds, Funder: Wolfson College Cambridge Junior Research Fellowship, Liquid–liquid phase‐separated biomolecular systems are increasingly recognized as key components in the intracellular milieu where they provide spatial organization to the cytoplasm and the nucleoplasm. The widespread use of phase‐separated systems by nature has given rise to the inspiration of engineering such functional systems in the laboratory. In particular, reversible gelation of liquid–liquid phase‐separated systems could confer functional advantages to the generation of new soft materials. Such gelation processes of biomolecular condensates have been extensively studied due to their links with disease. However, the inverse process, the gel–sol transition, has been less explored. Here, a thermoresponsive gel–sol transition of an extracellular protein in microgel form is explored, resulting in an all‐aqueous liquid–liquid phase‐separated system with high homogeneity. During this gel–sol transition, elongated gelatin microgels are demonstrated to be converted to a spherical geometry due to interfacial tension becoming the dominant energetic contribution as elasticity diminishes. The phase‐separated system is further explored with respect to the diffusion of small particles for drug‐release scenarios. Together, this all‐aqueous system opens up a route toward size‐tunable and monodisperse synthetic biomolecular condensates and controlled liquid–liquid interfaces, offering possibilities for applications in bioengineering and biomedicine.