Biology subjects

Buell, A. K.

Publications and source records attributed to Buell, A. K..

18 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2026

Structural thermodynamics by cryo-EM resolves minute energy differences in heterogeneous protein complexes.

Hetero-oligomeric protein complexes play crucial roles across biology, providing combinatorial diversity and context-specific functionality in membrane receptors, molecular chaperones and beyond. Yet, existing methods are limited in their ability to resolve and quantify the assembly energetics of complex biomolecular populations. Here, we introduce a comprehensive method combining iterative 3D classification, error-rate quantification via simulated particles, and global thermodynamic modeling to extract interaction energies from cryo-EM data. Using hexameric cruciferin (the primary rapeseed storage protein) as a model, we reconstruct a 1.83 [A] structure and distinguish three highly similar 50 kDa isoforms. We resolve the complex compositional heterogeneity of the hexameric species at the single-particle level and fit a global binding model revealing pairwise interaction energies between isoforms differing by fractions of the thermal energy (kB T ). This method offers a powerful, broadly applicable tool to characterize the compositional heterogeneity and energetics of biomolecular complexes.

biophysics↗

bioRxiv2026

De novo Design of Polymorph-Specific Binders Targeting α-Synuclein Fibrils

Alpha-synuclein (aSyn) aggregation into amyloid fibrils is a hallmark of synucleinopathies, a group of neurodegenerative disorders that includes Parkinsons disease. Beyond the central role of fibrils in disease pathology and propagation, distinct aSyn fibril polymorphs have been associated with different synucleinopathies, presenting opportunities for differential diagnosis and structure-based disease-specific phenotyping. Here, we present AmyBind, a computational pipeline for the de novo design of mini-protein binders that target polymorph-specific structural features of amyloid fibrils. Using this approach, we developed two aSyn fibril-specific binders, one of which exhibited polymorph-specificity. Neither binder interfered with fibril elongation in vitro, consistent with their designed binding mode on lateral fibril surfaces. In cell-based models, both binders showed polymorph-specific colocalization but did not affect fibril uptake or seeding, indicating their utility as fibril-selective labeling agents in cellular contexts. These findings demonstrate the feasibility of structure-guided, polymorph-specific amyloid targeting and provide a foundation for developing early-stage diagnostics and therapeutics for synucleinopathies and other amyloid-related diseases.

bioengineering↗

bioRxiv2025

Probing the energy landscape of α-Synuclein amyloid fibril formation by systematic K-to-Q mutagenesis

The aggregation of natively disordered -Synuclein (Syn) into amyloid fibrils is a hallmark of Parkinsons and other neurodegenerative diseases. Understanding Syns pathological role remains a major challenge due to its complex, context-dependent energy landscape characterized by conformational plasticity and fibril polymorphism. Here, we present a systematic mutational analysis as a quantitative probe of the Syn energy landscape, focusing on electrostatic contributions to key aggregation pathways. We engineered Syn variants with one to eight lysine-to-glutamine substitutions and analyzed their aggregation under controlled conditions to delineate their effects on nucleation, elongation, seed amplification, fibril stability, and fibril polymorphism. We find that Syn aggregation from a homogenous solution can be modelled well using global properties, including protein concentration, charge, and ionic strength. Microscopic pathways and the resulting fibril polymorphs are instead modulated by sequence-specific effects. We identify mutations of residues found in fibril cores as perturbations that significantly modify the Syn free energy landscape, creating pathways and energy minima not accessible to the WT under the same experimental conditions. In contrast, mutations outside of the fibril core affect the magnitude of the relevant energy barriers whilst overall maintaining a WT-like free energy landscape. Our work outlines a scalable, quantitative framework that increases the informational output of the mutational studies of Syn using conventional assays. The approach can be extended by incorporating additional mutational and functional data to deepen our understanding of Syns energy landscape and its role in health and disease.

biophysics↗

bioRxiv2025

Mechanism-Guided Engineering of Fluorinase Unlocks EfficientNucleophilic Biofluorination

The fluorinase enzyme, the only known biocatalyst forming stable carbon-fluorine bonds, operates with extremely low efficiency, catalyzing one reaction every 2-12 minutes. This severely limits its utility for sustainable biofluorination, and its sluggish activity remains poorly understood. We suppressed its aggregation through directed mutagenesis and elucidated the kinetic mechanism using a novel mathematical framework that fits complex kinetic and oligomerization data. This analysis revealed that >80% of enzyme molecules are inactive under standard conditions due to two dead-end pathways. The designed W50F+A279R mutant preferentially formed hexamers and displayed enhanced catalytic efficiency in this oligomeric state. When coupled with mechanism-based optimization of the reaction medium, including enzymatic removal of the inhibitory product, the catalytic turnover rate reached 12.5 {+/-} 2.1 min-{superscript 1}, representing [~]60-fold increase compared with previously reported turnover rates of the wild-type enzyme. Our work provides a mechanistic blueprint for fluorinase enhancement and a generalizable mathematical framework for analyzing kinetics of multimeric enzymes.

biochemistry↗

bioRxiv2025

RNA Selectively Modulates Activity of Virulent Amyloid PSMα3 and Host Defense LL-37 via Phase Separation and Aggregation Dynamics

Amyloid-forming peptides are increasingly recognized as dynamic regulators at the host-pathogen interface, yet how environmental factors control their assembly and activity remains poorly understood. Here, RNA acts as a concentration-dependent regulator of two sequence-related -helical peptides with fundamentally different assembly behaviors: the cross- amyloid-forming Staphylococcus aureus virulence factor PSM3 and the non-amyloidogenic human host-defense peptide LL-37. RNA drives PSM3 through distinct assembly states, from liquid-like condensates to fibrillar polymorphs, while preserving cytotoxic and antimicrobial activity over time. In contrast, RNA attenuates LL-37 cytotoxicity toward host cells while maintaining antibacterial activity, consistent with a host-protective immunomodulatory effect. Together with the opposing effects of EGCG, which redirects both peptides into amorphous assemblies, these findings support a mechanistic model in which biological activity is governed by supramolecular architecture, assembly trajectory, and dynamics rather than by monomer abundance or mature fibrils alone. More broadly, our findings identify RNA as an environmental regulator of -helical peptide assemblies and establish assembly-state control as a tunable determinant of virulence and host defense.

biophysics↗

bioRxiv2024

Thermodynamic Stability Modulates Chaperone-Mediated Disaggregation of α-Synuclein Fibrils

The aggregation of the intrinsically disordered protein alpha-synuclein into amyloid fibrils and their subsequent intracellular accumulation are characteristic features of several neurodegenerative disorders, such as Parkinsons disease. Currently, there are no curative treatment options available. In this study, we demonstrate that the thermodynamic stability of alpha-synuclein fibrils is a crucial factor influencing the efficiency with which they are disaggregated by the human chaperone system comprising HSP70, DNAJB1, and Apg2. We quantify the increasing stability of alpha-synuclein fibrils formed under four different solution conditions over a three-month incubation period. The chaperone system effectively disaggregates three out of the four fibril types, with varying efficiencies that correlate with their thermodynamic stability. The fibrils exhibit differential sensitivities to chaperone-mediated depolymerization, suggesting that both structural features and thermodynamic stability contribute to the susceptibility of alpha-synuclein fibrils to chaperone disaggregation. Our findings thus reveal a connection between the thermodynamic stability of fibrils and their susceptibility to chaperone-mediated disaggregation.

biophysics↗

bioRxiv2024

Proteome-scale quantification of the interactions driving condensate formation of intrinsically disordered proteins

Intrinsically disordered protein regions facilitate cellular organization through phase separation into biomolecular condensates. However, the molecular interactions driving this process remain poorly understood because of experimental limitations. Here, we advance experimental throughput by several orders of magnitude by developing Condensate Partitioning by mRNA-Display (CPmD). The method allows analysis of partitioning of hundred thousand peptides derived from the disordered proteome into reconstituted condensates. Our results demonstrate that the amino acid content, rather than specific sequence, primarily determines partitioning behavior. Importantly, quantification of the partitioning energies of peptides allows us to decipher the molecular grammar of the relevant interactions, allowing accurate prediction of the formation of condensates of diverse full-length disordered protein regions. The results reveal how physicochemical properties of disordered regions encode biological functions through formation of biomolecular condensates.

biophysics↗

bioRxiv2024

Structural polymorphism of alpha-synuclein fibrils alters pathway of Hsc70 mediated disaggregation

The pathological aggregation of -synuclein into amyloid fibrils is a hallmark of synucleinopathies including Parkinsons disease. Despite this commonality, synucleinopathies display divergent disease phenotypes that have been attributed to disease specific three-dimensional structures of -synuclein fibrils, each with a unique toxic gain-of-function profile. The Hsc70 chaperone is remarkable in its ability to disassemble pre-existing amyloid fibrils of different proteins in an ATP and co-chaperone dependent manner. We find however, using six well-defined conformational polymorphs of -synuclein fibrils, that the activity of the Hsc70 disaggregase machinery is sensitive to differences in the amyloid conformation, confirming that fibril polymorphism directly affects interactions with the proteostasis network. Amyloid conformation influences not only how efficiently fibrils are cleared by the Hsc70 machinery but also the preferred pathway of disaggregation. We further show that, in vitro, the active processing of fibrils by the Hsc70 machinery inadvertently produces seeding competent species that further promote protein aggregation. Amyloid conformation thus is an important feature that can tilt the balance between beneficial or detrimental protein quality control activities in the context of disease.

biochemistry↗

bioRxiv2024

Divergent effects of pathological α-synuclein truncations and mutations on phase separation

Phase separated condensates can accelerate -synuclein (-Syn) amyloid fibril formation implicated in Parkinsons disease pathogenesis. The effects of pathological modifications, i.e., truncations and familial mutations on the thermodynamics, material properties, and the extent of amyloid aggregation within -Syn condensates remain elusive. Here, we quantitatively demonstrate that terminal truncations significantly alter -Syn phase separation, while familial mutations impart minimal effects. Spontaneous sol-gel phase transitions of the truncated -Syn variants could give rise to amyloid fibrils almost instantly within condensates, suggesting similarities between molecular interactions driving both processes. Extending our study to model coacervate and condensate systems where -Syn acts as a client, we find -Syn can dissolve coacervates and form Pickering clusters on condensate surfaces--regulating their size. Additionally, the C-terminal region of -Syn modulates nucleic acid sequestration within condensates. Together, our findings reveal diverse effects of -Syn modifications on phase separation, both in pathological and physiological contexts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/624073v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1760f9dorg.highwire.dtl.DTLVardef@1fb155forg.highwire.dtl.DTLVardef@138402dorg.highwire.dtl.DTLVardef@1fd5229_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Global kinetic model of lipid-induced alpha-synuclein aggregation and its inhibition by small molecules

The aggregation of -synuclein into amyloid fibrils is a hallmark of Parkinsons disease. This process has been shown to directly involve interactions between proteins and lipid surfaces when the latter are present. Despite this importance, the molecular mechanisms of lipid-induced amyloid aggregation have remained largely elusive. Here, we present a global kinetic model to describe lipid-induced amyloid aggregation of -synuclein. Using this framework we find that -synuclein fibrils form via a two-step primary nucleation mechanism and that lipid molecules are directly involved in both the nucleation and fibril elongation steps, giving rise to lipid-protein coaggregates. To illustrate the applicability of this kinetic approach to drug discovery, we identify the mechanism of action of squalamine, a known inhibitor of -synuclein aggregation, finding that this small molecule reduces the rate of lipid-dependent primary nucleation. Our work will likely guide the rational design of -synuclein aggregation inhibitors. Significance StatementAmyloid aggregation is a hallmark of a diverse range of diseases including Parkinsons Disease, where the protein -synuclein is a major con-stituent of proteinaceous deposits found in patients. It is well established that interactions between -synuclein and lipids modulate aggregation. However, the molecular mechanisms driving this lipid-induced aggregation have remained largely elusive, which has frustrated so far the discovery of drugs that prevent lipid-induced aggregation. In this work, we present the first global kinetic model describing lipid-induced aggregation and by integrating this theoretical framework with in vitro experimental data of lipid-induced -synuclein aggregation, we reveal the role of lipid membranes in the aggregation process and uncover the mechanism by which small molecule inhibitors interfere with this process.

biophysics↗

bioRxiv2023

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Engineering improved B-family DNA polymerases to incorporate 3'-O-modified nucleotide reversible terminators is limited by an insufficient understanding of the structural determinants that define polymerization efficiency. To explore the key mechanism for unnatural nucleotide incorporation, we engineered a B-family DNA polymerase from Thermococcus Kodakaraenis (KOD pol) by using semi-rational design strategies. We first scanned the active pocket of KOD pol through site-directed saturation mutagenesis and combinatorial mutations and identified a variant Mut_C2 containing five mutation sites (D141A, E143A, L408I, Y409A, A485E) using a high-throughput microwell-based screening method. Mut_C2 demonstrated high catalytic efficiency in incorporating 3-O-azidomethyl-dATP labeled with a Cy3 dye, whereas the wild-type KOD pol failed to incorporate it. Computational simulations were then conducted towards the DNA binding region of KOD pol to predict additional mutations with enhanced catalytic activity, which were subsequently experimentally verified. By a stepwise combinatorial mutagenesis approach, we obtained an eleven-mutation variant, named Mut_E10 by introducing additional mutations to the Mut_C2 variant. Mut_E10, which carried six specific mutations (S383T, Y384F, V389I, V589H, T676K, and V680M) within the DNA-binding region, demonstrated over 20-fold improvement in kinetic efficiency as compared to Mut_C2. In addition, Mut_E10 demonstrated satisfactory performance in two different sequencing platforms (BGISEQ-500 and MGISEQ-2000), indicating its potential for commercialization. Our study demonstrates that an effective enhancement in its catalytic efficiency towards modified nucleotides can be achieved efficiently through combinatorial mutagenesis of residues in the active site and DNA binding region of DNA polymerase. These findings contribute to a comprehensive understanding of the mechanisms that underlie the incorporation of modified nucleotides by DNA polymerase. The beneficial mutation sites, as well as the nucleotide incorporation mechanism identified in this study, can provide valuable guidance for the engineering of other B-family DNA polymerases.

bioengineering↗

bioRxiv2022

Mass photometric detection and quantification of nanoscale α-synuclein phase separation

-Synuclein (-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinsons disease pathogenesis. Critical concentrations of -Syn LLPS are relatively high under physiological solution conditions. Moreover, -Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that -Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of molecules-- both above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for -Syn LLPS samples containing nanoscale droplets--which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates -Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of -Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding -Syn LLPS within physiological scales. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/490467v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@b355org.highwire.dtl.DTLVardef@1fdc4bdorg.highwire.dtl.DTLVardef@17f8aa8org.highwire.dtl.DTLVardef@682598_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Rational evolution of a recombinant DNA polymerase for efficient incorporation of unnatural nucleotides by dual-site boosting

Machine learning modelling assisting function-oriented enzyme engineering is normally built on predefined protein sequence space. However, efficient defining the determinant amino acid positions upon which the combinatorial mutation library is constructed is still a challenge in protein science. Herein, we present a comprehensive investigation of modifying a recombinant DNA polymerase for efficient incorporating one unnatural nucleotide, including the identification of key sites/regions, machine learning-assisted mutants screening, and the underlying mechanism of kinetics boosting. By using hundreds of training points and only dozens of testing samples, we found that one highly engineered enzymes catalytic efficiency can be further improved by one order of magnitude by specific mutation on two sites, 485I and 451L. Compared to the position 485 which is known to dominate local conformation of B-family DNA polymerases, 451 is a split-new active site discovered by our approach. A novel allosteric regulation mechanism is underlying the apparent synergy of 485I and 451L on the kinetics boosting. As a result, a "half-closed" conformation of the binding pocket and a cooperative binding of both primer and template DNA strands on the protein accelerated the processes of substrates incorporation, molecular recognition, and releasing of incorrect nucleotides. These findings have implications in guiding the function-tuning of DNA polymerases for a broad range of biotechnological applications.

biochemistry↗

bioRxiv2021

Universal amyloidogenicity of patient-derivedimmunoglobulin light chains

The deposition of immunoglobulin light chains (IgLCs) in the form of amorphous aggregates or amyloid fibrils in different tissues of patients can lead to severe and potentially fatal organ damage, requiring transplantation in some cases. There has been great interest in recent years to elucidate the origin of the very different in vivo solubilities of IgLCs, as well as the molecular determinants that drive either the formation of ordered amyloid fibrils or disordered amorphous aggregates. It is commonly thought that the reason of this differential aggregation behaviour is to be found in the amino acid sequences of the respective IgLCs, i.e. that some sequences display higher intrinsic tendencies to form amyloid fibrils. Here we perform in depth Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip) of 9 multiple myeloma patient-derived IgLCs, the amino acid sequences of all of which we have solved by de novo protein sequencing with mass spectrometry. The latter technique was also used for one IgLc from a patient with AL amyloidosis. We find that all samples also contain proteases that fragment the proteins under physiologically relevant mildly acidic pH conditions, leading to amyloid fibril formation in all cases. Our results suggest that while every pathogenic IgLC has a unique ThAgg fingerprint, all sequences have comparable amyloidogenic potential. Therefore, extrinsic factors, in particular presence of, and susceptibility to, proteolytic cleavage is likely to be a strong determinant of in vivo aggregation behaviour. The important conclusion, which is corroborated by systematic analysis of our sequences, as well as many sequences of IgLCs from amyloidosis patients reported in the literature, challenges the current paradigm of the link between sequence and amyloid fibril formation of pathogenic light chains.

biophysics↗

bioRxiv2020

Monomeric amyloid beta-peptide (1-42) significantly populates compact fibril-like conformations

The aggregation of the amyloid {beta} (A{beta}) peptide is a major hallmark of Alzheimers disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. The structure of the A{beta} monomer has been shown to be very dynamic and mostly disordered, both in experimental and in computational studies, similar to a random coil. This structural state of the monomer contrasts with the very stable and well defined structural core of the amyloid fibrils. An important question is whether the monomer can adopt transient fibril-like conformations in solution and what role such conformations might play in the aggregation process. Here we use enhanced and extensive molecular dynamics simulations to study the A{beta}42 monomer structural flexibility with different force fields, water models and salt concentrations. We show that the monomer behaves as a random coil under different simulation conditions. Importantly, we find a conformation with the N-terminal region structured very similarly to that of recent experimentally determined fibril models. This is to the best of our knowledge the first monomeric structural ensemble to show such a similarity with the fibril structure.

biophysics↗

bioRxiv2019

An engineered monomer binding-protein for α-synucleinefficiently inhibits the proliferation of amyloid fibrils

Removing or preventing the formation of -synuclein aggregates is a plausible strategy against Parkinsons disease. To this end we have engineered the {beta}-wrapin AS69 to bind monomeric -synuclein with high affinity. In cultured cells, AS69 reduced the occurrence of -synuclein oligomers and of visible -synuclein aggregates. In flies, AS69 reduced -synuclein aggregates and the locomotor deficit resulting from -synuclein expression in neuronal cells. In a mouse model based on the intracerebral injection of pre-formed -synuclein seed fibrills (PFFs), AS69 co-injection reduced the density of dystrophic neurites observed three months later. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited auto-catalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69--synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition. These results represent a new paradigm that high affinity monomer binders can be strongly sub-stoichiometric inhibitors of nucleation processes.

bioengineering↗

bioRxiv2017

A structural and kinetic link between membrane association and amyloid fibril formation of α-Synuclein

The protein -Synuclein (S) is linked to Parkinsons disease through its abnormal aggregation, which is thought to involve an interplay between cytosolic and membrane-bound forms of S. Therefore, better insights into the molecular determinants of membrane association and their implications for protein aggregation may help deciphering the pathogenesis of Parkinsons disease. Following previous studies using micelles and vesicles, we present a comprehensive study of S interaction with phospholipid bilayer nanodiscs. Using a combination of NMR - spectroscopic and complementary biophysical as well as computational methods we structurally and kinetically characterize S interaction with defined stable planar membranes in a quantitative and site-resolved way. We probe the role of S acetylation as well as membrane charge, plasticity and available surface area in modulating S membrane binding modes and directly link these findings to their consequences for S amyloid fibril formation.

biophysics↗