Biology subjects

Knowles, T. P. J.

Publications and source records attributed to Knowles, T. P. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

biophysics↗

bioRxiv2023

Single-molecule digital sizing of proteins in solution

Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable calibration-free single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

biophysics↗

bioRxiv2023

Self-replication of Abeta42 aggregates occurs on small and isolated fibril sites

Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyse the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modelling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behaviour in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimers A{beta}42 amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A{beta}42 fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.

biophysics↗

bioRxiv2023

Condensate partitioning governs the mechanism of action of FUS phase separation modulators

Biomolecular phase separation plays a pivotal role in governing critical biological functions and arises from the collective interactions of large numbers of molecules. Characterising the underlying collective interactions of phase separation, however, has proven to be challenging with currently available tools. Here, we propose a general and easily accessible strategy to quantify collective interactions in biomolecular phase separation with respect to composition and energetics. By measuring the dilute phase concentration of one species only, we determine tie line gradients and free energy dominance as dedicated descriptors of collective interactions. We apply this strategy to dissect the role of salts and small molecules on phase separation of the protein fused in sarcoma (FUS). We discover that monovalent salts can display both exclusion from or preferential partitioning into condensates to either counteract charge screening or enhance non-ionic interactions. Moreover, we show that the common hydrophobic interaction disruptor 1,6-hexanediol inhibits FUS phase separation by acting as a solvation agent capable of expanding the protein polypeptide chain. Taken together, our work presents a widely applicable strategy that enables quantification of collective interactions and provides unique insights into the underlying mechanisms of condensate formation and modulation.

biophysics↗

bioRxiv2023

Modulating Nucleic Acid Phase Transitions as a Mechanism of Action for Cell-Penetrating Antimicrobial Peptides

Antimicrobial peptides (AMPs) are a vital part of the immune system that helps combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we have discovered that a significant number of AMPs have a strong tendency to form liquid-like condensates in the presence of nucleic acids, a process known as phase separation. We have demonstrated that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. We propose that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. These findings open up exciting possibilities for the development of antimicrobials that target nucleic acid phase transitions.

microbiology↗

bioRxiv2023

Non-specificity fingerprints for clinical stage antibodies in solution

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs, does not solely rely on optimal potency and safety, but also require good biophysical properties to ensure high developability potential. In particular, non-specific interactions are a key developability measure to monitor during discovery. Despite an increased focus on the detection of non-specific interactions, their physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterise a set of clinical stage mAbs and their interactions with commonly used non-specificity ligands to generate non-specificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to evaluate the contribution of avidity in non-specific binding by mAbs. Based on our findings, we propose a quantitative solution-based non-specificity score, which can be exploited in the development of biological therapeutics and more widely in protein engineering.

biophysics↗

bioRxiv2022

Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains

Neurodegenerative diseases, such as Alzheimers Disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low molecular weight aggregates play a key role in disease-associated toxicity. Within these aggregates, protofibrillar loop-like structures have been observed for a variety of amyloid systems and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils has largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterise amyloid ring structures derived from the brains of AD patients. We analyse the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. Furthermore, we show that these findings can be extended to several amyloid systems, giving a general framework relating the mechanical properties of assemblies and the conditions in which they can form loop structures. These results explain the diversity in the structures formed from protein aggregation and sheds light on the links between early forms of flexible ring-forming aggregates and their role in disease.

molecular biology↗

bioRxiv2022

Enhanced surface nano-analytics of transient biomolecular processes

The study of the physical and chemical properties of biomolecules enables the characterisation of fundamental molecular processes and mechanisms in health and disease. Bulk and single-molecule analytical methods provide rich information on biomolecules, but often require high concentrations and sample preparation away from physiologically relevant conditions. Here, we present the development and application of a lab-on-a-chip approach which combines rapid sample preparation, mixing and deposition to integrate with a range of nano-analytical methods in chemistry and biology, providing enhanced sensitivity and single molecule resolution. We demonstrate that this method empowers multidimensional study of heterogenous biomolecular systems in physiological buffers and concentrations over multiple length scales by nanoscopy and vibrational spectroscopy. We illustrate the capabilities of this platform by capturing and analysing the structural conformations of transient oligomeric species formed at the early stages of the self-assembly of -synuclein, which are associated with the onset of Parkinsons disease. TEASERMaintaining the heterogeneity and structural integrity of monomers and oligomers enables their quantitative study.

biophysics↗

bioRxiv2022

Bioprinting microporous functional living materials from protein-based core-shell microgels

Living materials bring together material science and biology to allow the engineering and augmenting of living systems with novel functionalities. Bioprinting promises accurate control over the formation of such complex materials through programmable deposition of cells in soft materials, but current approaches had limited success in fine-tuning cell microenvironments while generating robust macroscopic morphologies. Here, we address this challenge through the use of core-shell microgel ink to decouple cell microenvironments from the structural shell for further processing. Cells are microfluidically immobilized in the viscous core that can promote the formation of both microbial populations and mammalian cellular spheroids, followed by interparticle annealing to give covalently stabilized functional scaffolds with controlled microporosity. The results show that the core-shell strategy mitigates cell leakage while affording a favorable environment for cell culture. Furthermore, we demonstrate that different microbial consortia can be printed into scaffolds for a range of applications. By compartmentalizing microbial consortia in separate microgels, the collective bioprocessing capability of the scaffold is significantly enhanced, shedding light on strategies to augment living materials with bioprocessing capabilities.

bioengineering↗

bioRxiv2022

Self-consistent solution to the Flory-Huggins model and applications in protein liquid-liquid phase separation

A self-consistent analytical solution for binodal concentrations of the two-component Flory-Huggins model is derived. We show that this form extends the validity of the Ginzburg-Landau expansion away from the critical point to cover the whole phase space. Furthermore, this analytical solution reveals an exponential scaling law of the dilute phase binodal concentration as a function of interaction strength and chain length. We demonstrate explicitly the power of this approach by fitting experimental protein liquid-liquid phase separation boundaries to determine the effective chain length and the solute-solvent interaction energy. Moreover, we demonstrate that this strategy allows us to resolve the differences in the interaction energy contributions of individual amino acids. This analytical framework can serve as a new way to decode the protein sequence grammar for liquid-liquid phase separation.

biophysics↗

bioRxiv2022

A Kinetic Map of the Influence of Biomimetic Lipid Membrane Models on Aβ42 Aggregation

The aggregation of the amyloid {beta} peptide (A{beta}) is one of the major molecular hallmarks of Alzheimers disease. Although A{beta} deposits have been mostly observed extracellularly, various studies have reported the presence of also intracellular A{beta} assemblies. Because these intracellular A{beta} aggregates might play a role in the onset and progression of Alzheimers disease, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein (APP), from which A{beta} is derived by proteolytic cleavage. Since lipid bilayers have been shown to promote the aggregation of A{beta}, in this study we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of the 42-residue form of A{beta} (A{beta}42). By using small unilamellar vesicles modelling cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the endoplasmic reticulum (ER), and the Golgi apparatus, we show that A{beta}42 aggregation is inhibited by the ER and Golgi membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on A{beta} aggregation, and suggest the presence of an evolutionary optimization of lipid composition to prevent the intracellular aggregation of A{beta}.

biophysics↗

bioRxiv2022

Surface interaction patches link non-specific binding and phase separation of antibodies

Non-specificity is a key challenge in the successful development of therapeutic antibodies. The tendency for non-specific binding in antibodies is often difficult to reduce via judicious design and, instead, it is necessary to rely on comprehensive screening campaigns. A better understanding of the molecular origins that drive antibody non-specificity is therefore highly desirable in order to prevent non-specific off-target binding. Here, we perform a systematic analysis of the impact of surface patch properties on antibody non-specificity using a designer antibody library as a model system and DNA as a non-specificity ligand. Using an in solution microfluidics approach, we discover patches of surface hydrogen bonding to be causative of the observed non-specificity under physiological salt conditions and suggest them to be a vital addition to the molecular origins of non-specificity. Moreover, we find that a change in formulation conditions leads to DNA-induced antibody liquid-liquid phase separation as a manifestation of antibody non-specificity. We show that this behaviour is driven by a cooperative electrostatic network assembly mechanism enabled by mutations that yield a positively charged surface patch. Together, our study provides a direct link between molecular binding events and macroscopic liquid-liquid phase separation. These findings highlight a delicate balance between surface interaction patches and their crucial role in conferring antibody non-specificity.

biophysics↗

bioRxiv2022

Multiphase condensates from a kinetically arrested phase transition

The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

biophysics↗

bioRxiv2022

Antibody affinity and concentration of convalescent sera provide context for reduced SARS-CoV-2 Omicron spike affinity of therapeutic antibodies

We assessed the affinities of the therapeutic monoclonal antibodies (mAbs) cilgavimab, tixagevimab, sotrovimab, casirivimab, and imdevimab to the receptor binding domain (RBD) of wild type, Delta, and Omicron spike. The Omicron RBD affinities of cilgavimab, tixagevimab, casirivimab, and imdevimab decreased by at least two orders of magnitude relative to their wild type equivalents, whereas sotrovimab binding was less severely impacted. These affinity reductions correlate with reduced antiviral activities of these antibodies, suggesting that simple affinity measurements can serve as an indicator for activity before challenging and time-consuming virus neutralization assays are performed. We also compared the properties of these antibodies to serological fingerprints (affinities and concentrations) of wild type RBD specific antibodies in 74 convalescent sera. The affinities of the therapeutic mAbs to wild type and Delta RBD were in the same range as the polyclonal response in the convalescent sera indicative of their high antiviral activities against these variants. However, for Omicron RBD, only sotrovimab retained affinities that were within the range of the polyclonal response, in agreement with its high activity against Omicron. Serological fingerprints thus provide important context to affinities and antiviral activity of mAb drugs and could guide the development of new therapeutics.

biochemistry↗

bioRxiv2021

Single-molecule sizing through nano-cavity confinement

An approach relying on nano-cavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nano-cavity diffusional sizing (NDS), measures particle residence times within fluidic nano-cavities to determine their hydrodynamic radii. Using theoretical modeling and simulation, we show that the residence time of particles within nano-cavities above a critical timescale depends on the diffusion coefficient of the particle, which allows estimation of the particles size. We demonstrate this approach experimentally through measurement of particle residence times within nano-fluidic 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 application in nanobiotechnology, biophysics, and clinical diagnostics. Table of content graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/472369v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abf7eorg.highwire.dtl.DTLVardef@d2180dorg.highwire.dtl.DTLVardef@1290f57org.highwire.dtl.DTLVardef@ae2087_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Structure-based discovery of small molecule inhibitors of the autocatalytic proliferation of alpha-synuclein aggregates

The presence of amyloid fibrils of -synuclein is closely associated with Parkinsons disease and related synucleinopathies. It is still very challenging, however, to systematically discover small molecules that prevent the formation of these aberrant aggregates. Here, we describe a structure-based approach to identify small molecules that specifically inhibit the surface-catalyzed secondary nucleation step in the aggregation of -synuclein by binding to the surface of the amyloid fibrils. The resulting small molecules are screened using a combination of kinetic and thermodynamic assays for their ability to bind -synuclein fibrils and prevent the further generation of toxic oligomers. This study demonstrates that the combination of structure-based and kinetic-based drug discovery methods can lead to the identification of small molecules that selectively inhibit the autocatalytic proliferation of -synuclein aggregates.

biophysics↗

bioRxiv2021

2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

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, -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.

biophysics↗

bioRxiv2021

Intermolecular reorganisation of single-componentcondensates during ageing promotes multiphasearchitectures.

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 StatementBiomolecular 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.

biophysics↗