scholarly journals Structure and dynamics underlying elementary ligand binding events in human pacemaking channels

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Marcel P Goldschen-Ohm ◽  
Vadim A Klenchin ◽  
David S White ◽  
John B Cowgill ◽  
Qiang Cui ◽  
...  

Although molecular recognition is crucial for cellular signaling, mechanistic studies have relied primarily on ensemble measures that average over and thereby obscure underlying steps. Single-molecule observations that resolve these steps are lacking due to diffraction-limited resolution of single fluorophores at relevant concentrations. Here, we combined zero-mode waveguides with fluorescence resonance energy transfer (FRET) to directly observe binding at individual cyclic nucleotide-binding domains (CNBDs) from human pacemaker ion channels critical for heart and brain function. Our observations resolve the dynamics of multiple distinct steps underlying cyclic nucleotide regulation: a slow initial binding step that must select a 'receptive' conformation followed by a ligand-induced isomerization of the CNBD. X-ray structure of the apo CNBD and atomistic simulations reveal that the isomerization involves both local and global transitions. Our approach reveals fundamental mechanisms underpinning ligand regulation of pacemaker channels, and is generally applicable to weak-binding interactions governing a broad spectrum of signaling processes.

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vojtech Vyklicky ◽  
Cherise Stanley ◽  
Chris Habrian ◽  
Ehud Y. Isacoff

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.


2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard

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.


2018 ◽  
Author(s):  
Alexander Carl DeHaven

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.


Author(s):  
Hsin-Chih Yeh ◽  
Christopher M. Puleo ◽  
Yi-Ping Ho ◽  
Tza-Huei Wang

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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