scholarly journals Detecting Intramolecular Dynamics and Multiple Förster Resonance Energy Transfer States by Fluorescence Correlation Spectroscopy

2010 ◽  
Vol 114 (17) ◽  
pp. 5895-5902 ◽  
Author(s):  
E. Shane Price ◽  
Matthew S. DeVore ◽  
Carey K. Johnson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Intramolecular Dynamics ◽  
Fluorescence Correlation

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

2021 ◽  
Author(s):  
Joanne C. Clark ◽  
Raluca A. I. Neagoe ◽  
Malou Zuidscherwoude ◽  
Deirdre M. Kavanagh ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Glycoprotein Vi ◽  
D2 Domain ◽  
Microscopy Techniques

AbstractCollagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

Single-Molecule Resonance Energy Transfer and Fluorescence Correlation Spectroscopy of Calmodulin in Solution†

2004 ◽  
Vol 108 (29) ◽  
pp. 10388-10397 ◽  
Author(s):  
Brian D. Slaughter ◽  
Michael W. Allen ◽  
Jay R. Unruh ◽  
Ramona J. Bieber Urbauer ◽  
Carey K. Johnson
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Visualization of Dysferlin Self-Interaction Using Fluorescence Resonance Energy Transfer and Fluctuation Correlation Spectroscopy

2011 ◽  
Vol 100 (3) ◽  
pp. 346a
Author(s):  
Renzhi Han ◽  
Li Xu ◽  
Sandeep Pallikkuth ◽  
Zhanjia Hou ◽  
Seth L. Robia
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Resonance

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.

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