Biology subjects

Gaub, H. E.

Publications and source records attributed to Gaub, H. E..

6 recordsLinked to original sources

bioRxiv2023

Effects of Variants of Concern Mutations on the Force-Stability of the SARS-CoV-2:ACE2 Interface and Virus Transmissibility

Viruses mutate under a variety of selection pressures, allowing them to continuously adapt to their hosts. Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to host factors, in particular to the cellular receptor ACE2. However, in the dynamic environment of the respiratory tract forces act on the binding partners, which raises the question whether not only affinity, but also force-stability of the SARS-CoV-2:ACE2 bond, might be a selection factor for mutations. Here, we use magnetic tweezers (MT) to study the effect of amino acid substitutions in variants of concern (VOCs) on RBD:ACE2 bond kinetics with and without external load. We find higher affinity for all VOCs compared to wt, in good agreement with previous affinity measurements in bulk. In contrast, Alpha is the only VOC that shows significantly higher force stability compared to wt. Investigating the RBD:ACE2 interactions with molecular dynamics simulations, we are able to rationalize the mechanistic molecular origins of this increase in force-stability. Our study emphasizes the diversity of contributions to the assertiveness of variants and establishes force-stability as one of several factors for fitness. Understanding fitness-advantages opens the possibility for prediction of likely mutations allowing rapid adjustment of therapeutics, vaccination, and intervention measures.

biophysics↗

bioRxiv2021

A Tethered Ligand Assay to Probe SARS-CoV-2:ACE2 Interactions

SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.

biophysics↗

bioRxiv2021

Kinetic Interval Measurement: A Tool to Characterize Thermal Reversion Dynamics of Light-switchable Fluorescent Proteins

Light-switchable proteins like Light-Oxygen-Voltage (LOV) domains can be used to control protein interactions and have been applied in vivo to manipulate cell behavior. The switching between dark and light state of LOV domains depends on temperature or their chemical microenvironment and can be tuned by point mutations. Here, we present a method called Kinetic Interval Measurement (KIM) to quantify the thermal reversion dynamics of light-switchable proteins by using a custom microplate reader. We show that this versatile method can be used to determine the reversion half-life of the excited state of LOV proteins in a reproducible, fast and simple manner consuming only small amounts of protein. The sensitivity of the method allows to report on changes in temperature and imidazole concentration as well as the photoswitching dynamics of LOV proteins in living cells.

biophysics↗

bioRxiv2020

A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

biophysics↗

bioRxiv2019

Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

Mechanical force can play a pivotal role in biological systems. Single Molecule Force Spectroscopy, is a powerful tool to probe the mechanics of proteins and their binding partners. Yet, it remains unclear how complex dissociation of a protein-protein interaction under mechanical forces occurs. Are receptor and ligand unbinding, or are they unfolding? We utilize an approach wherein receptor and ligand are expressed as a single molecule fused by a long flexible linker. Force is applied to the complex via an ultrastable handle. Consequently, the events during and following complex dissociation can be monitored. We investigate two high-affinity systems: The cohesin-dockerin type I interaction in which we find that a binding partner unfolds upon complex dissociation, and a colicin-immunity protein complex in which both proteins unfold completely upon unbinding. Mechanical receptor ligand dissociation thus can encompass unfolding of one or both binding partners.

biophysics↗

bioRxiv2018

Monomeric streptavidin: a versatile regenerative handle for force spectroscopy

Most avidin-based handles in force spectroscopy are tetravalent biotin binders. Tetravalency presents two issues: multiple pulling geometries as well as multiple targets bound simultaneously. Additionally, such tetravalent handles require elaborate purification protocols in order to reassemble. A stoichiometric, monomeric variant of streptavidin (mcSA2) had been engineered previously. It is readily expressed and purified, and it binds biotin with a nanomolar KD. For atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we fused the monomeric streptavidin with a small protein domain as an experimental fingerprint and to improve solubility. A ybbR-tag was additionally included for covalent site-specific tethering. Rupture forces of the mcSA2:biotin complex were found to be in a comparable range above 150 pN at force loading rates of 1E4 pN/s as for previously published, tetravalent streptavidin:biotin systems. Additionally, when tethering mcSA2 from its C-terminus, rupture forces were found to be slightly higher than when tethered N-terminally. Due to its monomeric nature, mcSA2 could also be chemically denatured and subsequently refolded - and thus regenerated during an experiment, in case the handle gets misfolded or clogged. We show that mcSA2 features a straightforward expression and purification with flexible tags, high stability, regeneration possibilities and an unambiguous pulling geometry. Combined, these properties establish mcSA2 as a reliable handle for single-molecule force spectroscopy.

biophysics↗