scholarly journals Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

2019 ◽  
Author(s):  
Lukas F. Milles ◽  
Hermann E. Gaub
Keyword(s):  
Single Molecule ◽  
Type I ◽  
Mechanical Forces ◽  
Single Molecule Force Spectroscopy ◽  
Receptor Ligand ◽  
Binding Partner ◽  
Ligand Dissociation ◽  
Protein Protein Interaction ◽  
Binding Partners ◽  
Mechanical Receptor

ABSTRACTMechanical 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.

scholarly journals Biomechanical Characterization of SARS-CoV-2 Spike RBD and Human ACE2 Protein-Protein Interaction

2020 ◽  
Author(s):  
Wenpeng Cao ◽  
Chuqiao Dong ◽  
Seonghan Kim ◽  
Decheng Hou ◽  
Wanbo Tai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Cell Types ◽  
Single Molecule Force Spectroscopy ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Loading Rates ◽  
Protein Protein Interaction ◽  
Biophysical Mechanism ◽  
The Difference

The current COVID-19 pandemic has already had a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use its surface spike (S) protein's receptor binding domain (RBD) to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD–ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS epidemic. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD–ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD–ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.

Single-molecule force spectroscopy on polyproteins and receptor–ligand complexes: The current toolbox

2017 ◽  
Vol 197 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Wolfgang Ott ◽  
Markus A. Jobst ◽  
Constantin Schoeler ◽  
Hermann E. Gaub ◽  
Michael A. Nash
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Receptor Ligand

A Combined In Vitro and In Silico Approach to Estimate the Molecular Arrangement Within a Fibronectin Fiber

2011 ◽  
Author(s):  
Mark J. Bradshaw ◽  
Michael L. Smith
Keyword(s):  
Single Molecule ◽  
Strain State ◽  
Cell Adhesion Molecule ◽  
Tertiary Structure ◽  
Cell Behavior ◽  
Single Molecule Force Spectroscopy ◽  
Binding Partner ◽  
Molecular Arrangement ◽  
Cellular Mechanotransduction

It has become apparent that the extracellular matrix (ECM) is a powerful modulator of cell behavior. Fibronectin (Fn) is of particular interest because it is a requisite cell adhesion molecule for development and wound healing and it is a promiscuous binding partner for many soluble signaling molecules. It was recently shown that the binding affinity of some molecules is dependent on the strain state of the Fn [2,3], reinvigorating our interest in the molecular mechanism of Fn fiber extension. The tertiary structure of the approximately 30 Fn type III domains of the protein has been shown to be capable of unfolding in single molecule force spectroscopy experiments, although evidence that unfolding occurs in Fn fibers has been indirect and has not been quantified. Nevertheless, unfolding of Fn molecules predicts a possible mechanism of strain dependant binding in Fn matrix and commensurate strain feedback to attached cells, contributing to the cellular mechanotransduction toolbox [3].

scholarly journals Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

2005 ◽  
Vol 169 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Daniela A. Sahlender ◽  
Rhys C. Roberts ◽  
Susan D. Arden ◽  
Giulietta Spudich ◽  
Marcus J. Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Interference ◽  
Vesicular Stomatitis Virus ◽  
Protein Interaction ◽  
Golgi Complex ◽  
Myosin Vi ◽  
Binding Partner ◽  
Binding Partners ◽  
Vesicular Stomatitis ◽  
Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

scholarly journals Single Molecule Force Spectroscopy of Siloxanes

2021 ◽  
Vol 714 (3) ◽  
pp. 032023
Author(s):  
Ling Chen ◽  
Liya Yang ◽  
Chunxia Wang ◽  
Ting Zhu
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

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