Biology subjects

Korosy, C.

Publications and source records attributed to Korosy, C..

5 recordsLinked to original sources

bioRxiv2026

CALISTO: A Standardized and Open-Source Software for Magnetic Tweezer Force Calibration

Magnetic tweezers (MT) enable multiplexed single-molecule force spectroscopy while applying controlled forces and torques to biomolecules. They resolve forces below 1 pN and maintain constant forces over extended periods, but accurate calibration remains challenging due to drift, tracking artifacts, camera noise, and bead variability. Here, we present CALISTO (CALIbration Suite for magnetic Tweezer Operation), an open-source software for MT force calibration. CALISTO analyzes position traces of DNA-tethered magnetic beads and surface-bound reference beads for drift correction. It implements three thermal-motion-based calibration methods: power spectral density, Allan variance, and Hadamard variance, with the latter providing increased robustness against drift and correlated measurement noise. The graphical user interface combines automated analysis with user-guided quality control, including reference bead selection, z-offset determination, magnet-position classification, bead-wise force estimation, and tether extension analysis. Together, these steps generate a master force calibration curve. We systematically evaluated their effects on calibration performance. The largest improvement resulted from automated filtering of problematic force plateaus using time-series characterization to identify traces affected by drift, tracking artifacts, or irregular fluctuations. Filtering reduced scatter and improved force estimation while preserving the underlying force-magnet position relationship. CALISTO thus provides a standardized, reproducible framework for reliable magnetic-tweezers force calibration.

biophysics↗

bioRxiv2026

Magnetic Tweezers Experiments Reveal Increased Mechanical Sensitivity of the F2561Y Mutant in Von Willebrand Factor

The large glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis. By sensing hydrodynamic forces in the bloodstream, VWF elongates under elevated shear, increasing its adhesiveness to platelets. This mechanosensitive behavior arises from its multidomain architecture, where distinct structural elements respond at different force scales. The stem, formed by interactions between C-domains of adjacent monomers in VWF dimers, opens at relatively low forces (~1 pN), representing an early step in force activation. The F2561Y mutation in the C4 domain is a gain-of-function mutation associated with increased shear sensitivity and myocardial infarction, yet its effect on VWF mechanics in the physiologically relevant low-force regime remains unresolved. Here, we combine single-molecule magnetic tweezers with AFM imaging to investigate how F2561Y alters VWF stem dynamics. Magnetic tweezers measurements reveal that A2 domain unfolding remains unchanged in the mutant, whereas stem opening transitions occur at significantly lower forces compared to wildtype. The midpoint force for stem opening is reduced by approximately one order of magnitude, indicating a pronounced destabilization of the compact stem conformation. AFM imaging independently confirms a higher population of open stem states in the mutant, even without applied force. Our findings demonstrate that F2561Y selectively destabilizes the VWF stem and increases its force sensitivity without perturbing global domain stability, providing a direct molecular explanation for its enhanced shear responsiveness and prothrombotic phenotype. More broadly, our work establishes magnetic tweezers as a powerful approach to resolve low-force conformational equilibria in mechanosensitive proteins and to dissect the mechanical impact of disease-associated mutations. HighlightsO_LIThe F2561Y mutation lowers the force required to open the VWF stem. C_LIO_LIF2561Y mutant VWF dimers exhibit more open conformations compared to wildtype. C_LIO_LIForce sensitivity provides a mechanism for the prothrombic phenotype of F2561Y. C_LI

biophysics↗

bioRxiv2025

The Effects of DMSO on DNA Conformations and Mechanics

Dimethyl sulfoxide (DMSO) is a polar aprotic solvent used in a wide range of applications, including uses as a drug and in drug delivery, as a solvent for fluorescence dyes, and in enzymatic reactions that process DNA. Consequently, many assays contain low concentrations ([≤] 10%) of DMSO. While it is well known that DMSO lowers the melting temperature of DNA, its effects on DNA conformations and mechanical properties below the melting temperature are unclear. Here we use complementary single-molecule techniques to probe DNA in the presence of 0-60% DMSO. Magnetic tweezers force-extension measurements find that the bending persistence length of DNA decreases moderately and linearly with DMSO concentrations up to 20 vol%, by (0.43 {+/-} 0.02)% per %-DMSO, respectively. Magnetic tweezers twist measurements demonstrate a reduction in melting torque in the presence of DMSO and find that the helical twist of DNA remains largely unchanged up to 20% DMSO, while even higher concentrations slightly unwind the helix. Using AFM imaging, we find a moderate compaction of DNA conformations by DMSO and observe a systematic decrease of the mean squared end-to-end distance by 1.2% per %-DMSO. We use coarse grained Monte Carlo simulations of DNA as a semi-flexible polymer with a variable density of flexible segments or bubbles, representing DMSO-induced local defects or melting, to rationalize the observed behavior. The model quantitates the effects of introducing locally flexible regions into DNA and gives trends in line with the magnetic tweezers and AFM imaging experiments. Our results show that addition of up to 50% DMSO has a gradual effect on DNA structure and mechanics and that for low concentrations ([≤] 20%) the induced changes are relatively minor. Our work provides a baseline to understand and model the effects of DMSO on DNA in a range of biophysical and biochemical assays. STATEMENT OF SIGNIFICANCEDimethyl sulfoxide (DMSO) is a widely used polar aprotic solvent. It is employed e.g. as a drug and in drug delivery, as a solvent for small molecules, and as an additive to enzymatic reactions that process DNA. Despite its wide-spread use, its effects on DNA conformations and properties are not well understood and often neglected. We use single-molecule manipulation with magnetic tweezers and AFM imaging to probe DNA in the presence of 0-60% DMSO. We find that DMSO increases the flexibility of DNA, leading to more compact conformations. Up to 50% DMSO, the induced changes are gradual and approximately linear. Our results provide a quantitative baseline to understand, model, and optimize assays with DNA in the presence of DMSO.

biophysics↗

bioRxiv2024

Accurate Drift-Invariant Single-Molecule Force Calibration Using the Hadamard Variance

Single-molecule force spectroscopy (SMFS) techniques play a pivotal role in unraveling the mechanics and conformational transitions of biological macromolecules under external forces. Among these techniques, multiplexed magnetic tweezers (MTs) are particularly well suited to probe very small forces, [≤]1 pN, critical for studying non-covalent interactions and regulatory conformational changes at the single-molecule level. However, to apply and measure such small forces, a reliable and accurate force calibration procedure is crucial. Here, we introduce a new approach to calibrate MTs based on thermal motion using the Hadamard variance (HV). To test our method, we develop a bead-tether Brownian dynamics simulation that mimics our experimental system and compare the performance of the HV method against two established techniques: power spectral density (PSD) and Allan variance (AV) analyses. Our analysis includes an assessment of each methods ability to mitigate common sources of additive noise, such as white and pink noise, as well as drift, which often complicate experimental data analysis. Our findings demonstrate that the HV method exhibits overall similar or even higher precision and accuracy, yielding lower force estimation errors across a wide range of signal-to-noise ratios (SNR) and drift speeds compared to the PSD and AV methods. Notably, the HV method remains robust against drift, maintaining consistent uncertainty levels across the entire studied SNR and drift speed spectrum. We also explore the HV method using experimental MT data, where we find overall smaller force estimation errors compared to PSD and AV approaches. Overall, the HV method offers a robust method for achieving sub-pN resolution and precision in multiplexed MT measurements. Its potential extends to other SMFS techniques, presenting exciting opportunities for advancing our understanding of mechano-sensitivity and force generation in biological systems. Therefore, we provide a well-documented Python implementation of the HV method as an extension to the Tweezepy package. Statement of Signi[fi]canceSingle-molecule force spectroscopy techniques are vital for studying the mechanics and conformations of bio-macromolecules under external forces. Multiplexed magnetic tweezers (MTs) excel in applying forces [≤] 1 pN, which are critical for examining non-covalent interactions and regulatory changes at the single-molecule level. Precise and reliable force calibration is essential for these measurements. In this study, we present a new force calibration method for multiplexed MTs using Hadamard variance (HV) based on thermal motion. The HV method shows similar or even higher precision and accuracy to established techniques like power spectral density and Allan variance. Most significantly, it is drift-invariant, maintaining consistent performance across varying experimental conditions. This robustness against drift ensures reliable force application and measurements at sub-pN resolution.

biophysics↗

bioRxiv2024

Determination of Absolute Intramolecular Distances in Proteins by Anomalous X-ray Scattering Interferometry

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

biophysics↗