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Monitoring of NBD-probes and their location in lipid membranes via their triplet state parameters
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimentell biomolekylär fysik)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

By a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST)imaging, the triplet state kinetics of the membrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl(NBD) was studied in small unilamellar vesicles (SUVs). It was shown that spin-labels, included inSUV membranes together with the NBD-labeled lipids, induce prominent effects on the triplet statekinetics of NBD. The relative effects on the triplet state kinetics are considerably larger than thoseobserved in traditional fluorescence quenching studies, and can provide information about thelocalization and the interactions between the lipids in the SUVs, using considerably lowerconcentrations of spin-labeled lipids in the membranes. From the effects of the spin labels on thetriplet state kinetics of NBD, we revisited the folding behavior of NBD-labeled phospholipid chains inthe membranes. Our results indicate that the NBD probe on the acyl chain of the phospholipids do notunambiguously loop back towards the membrane surface, as previously reported, but may alternatebetween a straight and a folded acyl chain, with the NBD label at the surface, or deep into themembrane bilayer. Our study suggests that the triplet state parameters of NBD can provide anadditional set of orthogonal parameters, which can increase accuracy and precision of fluorescencebasedmolecular dynamics and interaction studies with NDB as a probe molecule.

Keyword [en]
FCS, TRAST, lipid membrane, NBD
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-187739OAI: oai:DiVA.org:kth-187739DiVA: diva2:931464
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-27 Last updated: 2016-05-27Bibliographically approved
In thesis
1. Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Open this publication in new window or tab >>Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. x, 59 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2016:22
Keyword
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-187708 (URN)978-91-7729-026-1 (ISBN)
Public defence
2016-06-13, FB52, KTH, AlbaNova University Center, Roslagsvägen 30 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-26 Last updated: 2016-05-31Bibliographically approved

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Chmyrov, VolodymyrTornmalm, Johan
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