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Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimentell biomolekylär fysik)
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 [en]
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
National Category
Biophysics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-187708ISBN: 978-91-7729-026-1 (print)OAI: oai:DiVA.org:kth-187708DiVA: diva2:931216
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
List of papers
1. Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
Open this publication in new window or tab >>Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-107520 (URN)
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QS 2012

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2016-05-27Bibliographically approved
2. Highly Sensitive FRET-FCS Detects Amyloid beta-Peptide Oligomers in Solution at Physiological Concentrations
Open this publication in new window or tab >>Highly Sensitive FRET-FCS Detects Amyloid beta-Peptide Oligomers in Solution at Physiological Concentrations
2015 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 23, 11700-11705 p.Article in journal (Refereed) Published
Abstract [en]

Oligomers formed by the amyloid beta-peptide (A beta) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, A beta oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of FOrster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of A beta, FRET-FCS detected oligomers consisting of less than 10 A beta molecules, which coexisted with the monomers at fractions as low as 2 +/- 2%. Thus, we demonstrate for the first time direct detection of small fractions of A beta oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2015
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-180226 (URN)10.1021/acs.analchem.5b02630 (DOI)000365931100016 ()26489794 (PubMedID)2-s2.0-84948448366 (Scopus ID)
Note

QC 20160119

Available from: 2016-01-19 Created: 2016-01-08 Last updated: 2017-11-30Bibliographically approved
3. Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
Open this publication in new window or tab >>Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
Show others...
2014 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, 3254- p.Article in journal (Refereed) Published
Abstract [en]

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Place, publisher, year, edition, pages
Nature Publishing Group, 2014
Keyword
animal; biosynthesis; chemistry; dimerization; genetics; metabolism; nuclear magnetic resonance spectroscopy; pH; spectrofluorometry; spider; static electricity
National Category
Biochemistry and Molecular Biology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-187706 (URN)10.1038/ncomms4254 (DOI)000332667600001 ()
Funder
Danish National Research FoundationSwedish Research Council
Note

QC 20160527

Available from: 2016-05-26 Created: 2016-05-26 Last updated: 2017-11-30Bibliographically approved
4. Monitoring of NBD-probes and their location in lipid membranes via their triplet state parameters
Open this publication in new window or tab >>Monitoring of NBD-probes and their location in lipid membranes via their triplet state parameters
(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
FCS, TRAST, lipid membrane, NBD
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-187739 (URN)
Note

QC 20160527

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

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