A5043661581- Force Microscopy Techniques and Applications
- Mechanical and Optical Resonators
- Cellular Mechanics and Interactions
- Bacteriophages and microbial interactions
- SARS-CoV-2 and COVID-19 Research
- Molecular Junctions and Nanostructures
- RNA and protein synthesis mechanisms
- Viral Infections and Outbreaks Research
- Monoclonal and Polyclonal Antibodies Research
- Cell Adhesion Molecules Research
- Protein Structure and Dynamics
- COVID-19 Clinical Research Studies
- Cancer and biochemical research
- Influenza Virus Research Studies
- Advanced Biosensing Techniques and Applications
- Genomics and Phylogenetic Studies
- Erythrocyte Function and Pathophysiology
- Prion Diseases and Protein Misfolding
- Nanofabrication and Lithography Techniques
- Bacterial Genetics and Biotechnology
- Cardiomyopathy and Myosin Studies
- Management and Organizational Practices
- Social and Educational Sciences
- Biotin and Related Studies
- Biochemical and Structural Characterization
University of Washington
2024
Center for NanoScience
2015-2023
Stanford University
2022-2023
Ludwig-Maximilians-Universität München
2015-2023
LMU Klinikum
2022
Nanosystems Initiative Munich
2019
Center for Integrated Protein Science Munich
2017-2019
Yeshiva University
1995
Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal complexes formed at the cytoplasmic side of attachment to ECM activated after force generation via actomyosin fibers attached this complex. The mechanism translating mechanical biochemical signal not understood, it clear whether directly by or downstream signal. We use experimental computational single-molecule spectroscopy probe properties examine can trigger...
Significance In the dynamic environment of airways, where SARS-CoV-2 infections are initiated by binding to human host receptor ACE2, mechanical stability viral attachment is a crucial fitness advantage. Using single-molecule force spectroscopy techniques, we mimic effect coughing and sneezing, thereby testing RBD:ACE2 interaction under physiological conditions. Our results reveal higher ACE2 compared SARS-CoV-1, causing possible assay sensitive blocking agents preventing bond formation. It...
Proteins which bind intrinsically disordered proteins (IDPs) and regions (IDRs) with high affinity specificity could have considerable utility for therapeutic diagnostic applications. However, a general methodology targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of input, freely sampling both binding protein conformation, RFdiffusion can generate binders IDPs IDRs in wide range conformations. We use this approach Amylin, C-peptide VP48...
Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the has benefitted greatly from new approaches to bioconjugation of proteins surfaces. Indeed, optimized immobilization strategies for biomolecules refined purification schemes are being steadily adapted improved, which turn enhanced data quality. many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used anchor molecules interest...
Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on cell surface. Force-mediated dissociation of is thought to activate signaling, how this mechanosensitivity arises poorly understood. Here, we use magnetic tweezer assays show physiologically relevant...
Abstract G protein-coupled receptors (GPCRs) play key roles in physiology and are central targets for drug discovery development, yet the design of protein agonists antagonists has been challenging as GPCRs integral membrane proteins conformationally dynamic. Here we describe computational de novo methods a high throughput “receptor diversion” microscopy based screen generating GPCR binding miniproteins with affinity, potency selectivity, use these to generate MRGPRX1 CXCR4, GLP1R, GIPR,...
The widely used interaction of the homotetramer streptavidin with small molecule biotin has been intensively studied by force spectroscopy and become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse propagation pathways through different binding interfaces. This multiplicity gives rise to polydisperse data. Here, we present an engineered monovalent tetramer single cysteine its functional subunit that allows site-specific immobilization...
Since the development of green fluorescent protein, proteins (FP) are indispensable tools in molecular biology. Some FPs change their structure under illumination, which affects interaction with other biomolecules or proteins. In particular, that able to form switchable dimers became an important tool field optogenetics. They widely used for investigation signaling pathways, control surface recruitment, as well enzyme and gene regulation. However, optogenetics did not yet develop...
Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes connections cells establish between actomyosin cytoskeleton and extracellular matrix via integrins or to neighboring cadherins, yet little known regarding its design. binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs associate with vinculin head domain. We studied such complexes by pulling VBS peptides derived...
Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth myosin light chain kinase (smMLCK) is a member of diverse group serine/threonine kinases that feature association. Its catalytic activity triggered by conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK suspected be also regulatable mechanical stress. In this...
Abstract Most avidin-based handles in force spectroscopy are tetravalent biotin binders. Tetravalency presents two issues: multiple pulling geometries as well targets bound simultaneously. Additionally, such require elaborate purification protocols order to reassemble. A stoichiometric, monomeric variant of streptavidin (mcSA2) had been engineered previously. It is readily expressed and purified, it binds with a nanomolar K D . For atomic microscopy-based single-molecule (AFM-SMFS), we fused...
ABSTRACT The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. attaches to human host cells through interaction of its receptor binding domain (RBD) located on viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) surface cells. RBD ACE2 critical first step in infection. Viral attachment occurs dynamic environments where forces act partners multivalent interactions play central roles, creating an urgent need for assays that...
Abstract The mechanobiology of receptor–ligand interactions and force‐induced enzymatic turnover can be revealed by simultaneous measurements force response fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic (AFM). A fully automatized workflow is established manipulate single molecules inside ZMWs autonomously...
ABSTRACT SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on viral Spike protein to angiotensin-converting enzyme-2 (ACE2) human host cells. This critical first step occurs in dynamic environments, where external forces act binding partners and multivalent interactions play roles, creating an urgent need for assays that can quantitate with ACE2 under mechanical load defined geometries. Here, we introduce a tethered ligand assay comprises RBD ectodomain...
ABSTRACT Viruses mutate under a variety of selection pressures, allowing them to continuously adapt their hosts. Mutations in SARS-CoV-2 have shown effective evasion population immunity and increased affinity host factors, particular the cellular receptor ACE2. However, dynamic environment respiratory tract forces act on binding partners, which raises question whether not only affinity, but also force-stability SARS-CoV-2:ACE2 bond, might be factor for mutations. Here, we use magnetic...
Abstract Viruses mutate under a variety of selection pressures, allowing them to continuously adapt their hosts. Mutations in SARS-CoV-2 have shown effective evasion population immunity and increased affinity host factors, particular the cellular receptor ACE2. However, dynamic environment respiratory tract forces act on binding partners, which raises question whether not only affinity, but also force-stability SARS-CoV-2:ACE2 bond, might be factor for mutations. Here, we use magnetic...