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.

scholarly journals Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents

2019 ◽  
Vol 9 (1) ◽  
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 ◽  
Micron Size ◽  
Triton X ◽  
Kinetics Of

Abstract The 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.

scholarly journals Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

2004 ◽  
Vol 70 (8) ◽  
pp. 4561-4568 ◽  
Author(s):  
Jim O'Mahony ◽  
Colin Hill
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Triton X ◽  
Excellent Reproducibility

ABSTRACT Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg2+ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 106 to 3 × 101 copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.

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 Mechanism of DNA surface exploration and operator bypassing during target search

2020 ◽  
Author(s):  
E. Marklund ◽  
B. van Oosten ◽  
G. Mao ◽  
E. Amselem ◽  
K. Kipper ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Single Molecule ◽  
Time Scale ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Signal ◽  
Target Search ◽  
Fluorescence Correlation ◽  
Speed Accuracy

SummaryMany proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1–5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 μs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

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