scholarly journals CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

2018 ◽  
Author(s):  
C. Liesche ◽  
J. Berndt ◽  
F. Fricke ◽  
S. Aschenbrenner ◽  
M. Heilemann ◽  
...  
Keyword(s):  
Cell Death ◽  
Ligand Binding ◽  
Single Molecule ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Caspase 8 ◽  
List Type ◽  
Death Domain

AbstractCD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Förster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.HighlightsAt a density of less than 10 receptors per µm2CD95 exists as monomer (58%) and dimer (42%)Pre-formed dimers do not contribute to ligand-induced CD95 apoptotic signalingThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell deathsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitmentFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity

scholarly journals Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vojtech Vyklicky ◽  
Cherise Stanley ◽  
Chris Habrian ◽  
Ehud Y. Isacoff
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Detectable Effect ◽  
Open Probability ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Amino Terminal Domain

AbstractN-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

scholarly journals Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 103 ◽  
Author(s):  
Natasha D. Durham ◽  
Angela R. Howard ◽  
Ramesh Govindan ◽  
Fernando Senjobe ◽  
J. Maximilian Fels ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Transmembrane Protein ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Target Cells ◽  
Viral Fusion

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

scholarly journals Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Lu Zhang ◽  
Shaowen Wu ◽  
Yitao Feng ◽  
Dan Wang ◽  
Xilin Jia ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Binding Sites ◽  
Binding Protein ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Affinities ◽  
Binding Mechanism

AbstractProtein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

Towards Single-Molecule Diagnostics Using Microfluidic Manipulation and Quantum Dot Nanosensors

2007 ◽  
Author(s):  
Hsin-Chih Yeh ◽  
Christopher M. Puleo ◽  
Yi-Ping Ho ◽  
Tza-Huei Wang
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Detection ◽  
Specific Analysis ◽  
Low Volume

In this report, we review several single-molecule detection (SMD) methods and newly developed nanocrystal-mediated single-fluorophore strategies for ultrasensitive and specific analysis of genomic sequences. These include techniques, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis, which allow separation-free detection of low-abundance DNA sequences and mutational analysis of oncogenes. Microfluidic approaches developed for use with single-molecule detection to achieve rapid, low-volume, and quantitative analysis of nucleic acids, such as electrokinetic manipulation of single molecules and confinement of sub-nanoliter samples using microfluidic networks integrated with valves, are also discussed.

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