scholarly journals Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Suhaila Rajab ◽  
Leah Bismin ◽  
Simone Schwarze ◽  
Alexandra Pinggera ◽  
Ingo H. Greger ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Signal Transmission ◽  
Correlation Spectroscopy ◽  
Fluorescence Probes ◽  
Allosteric Coupling ◽  
Fluorescence Correlation ◽  
Binding Domains ◽  
Domain Motions ◽  
Kinetics Of

AbstractIonotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.

scholarly journals Unveiling the multi-step solubilization mechanism of single vesicles by detergents

2019 ◽  
Author(s):  
Paul A. Dalgarno ◽  
José Juan-Colás ◽  
Gordon J. Hedley ◽  
Lucas Piñeiro ◽  
Mercedes Novo ◽  
...  
Keyword(s):  
Real Time ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Optical Methods ◽  
Fluorescence Correlation ◽  
Ionic Detergent ◽  
Triton X ◽  
Kinetics Of ◽  
Single Vesicle

AbstractThe solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells

2001 ◽  
Vol 382 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Elke Van Craenenbroeck ◽  
Jo Vercammen ◽  
Gunther Matthys ◽  
Jan Beirlant ◽  
Christophe Marot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Serotonin Receptor ◽  
Specific Binding ◽  
Statistical Tests ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Escherichia Coli Cells ◽  
Multiple Binding Sites ◽  
Multiple Binding

Abstract A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore coated beads that mimic the behaviour of multiliganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Molecular Exchange Kinetics of Diblock Copolymer Micelles Monitored by Fluorescence Correlation Spectroscopy

2014 ◽  
Vol 3 (5) ◽  
pp. 428-432 ◽  
Author(s):  
David Schaeffel ◽  
Andreas Kreyes ◽  
Yi Zhao ◽  
Katharina Landfester ◽  
Hans-Jürgen Butt ◽  
...  
Keyword(s):  
Diblock Copolymer ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Exchange Kinetics ◽  
Copolymer Micelles ◽  
Kinetics Of

A fluorescence correlation spectroscopy study of ligand interaction with cytokinin-specific binding protein from mung bean

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Paweł Zawadzki ◽  
Genowefa Ślósarek ◽  
Jerzy Boryski ◽  
Przemysław Wojtaszek
Keyword(s):  
Ligand Binding ◽  
Diffusion Coefficients ◽  
Fluorescence Correlation Spectroscopy ◽  
Mung Bean ◽  
Specific Binding ◽  
Binding Protein ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Specific Binding Protein ◽  
Two Populations

Abstract Cytokinins are essential plant hormones that regulate numerous physiological processes. Recently, a protein was identified in mung bean (Vigna radiata) and characterized as a cytokinin-specific binding protein (VrCSBP). Fluorescence correlation spectroscopy was used to investigate the interaction between VrCSBP and its ligands. The synthetic cytokinin, N-phenyl-N′-(4-pyridyl) urea, was labeled with two fluorophores, 7-nitro-2,1,3-benzoxadiazole and rhodamine B. Protein-ligand binding was analyzed in an equilibrium saturation binding experiment and confirmed by the competition assay. Surprisingly, it was found that VrCSBP binds not only to cytokinins, but also to gibberellins. In addition, in the presence of natural cytokinins and gibberellins, two populations of VrCSBP that differ in their diffusion coefficients were detected. The diffusion coefficients of these two populations could be related to mono- and dimeric states, which suggests a new mode of operation in ligand binding by VrCSBP, in which dimerization induced by natural ligands enhances the ligand binding capacity of the protein.

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