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Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.ORCID-id: 0000-0002-6191-9921
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.ORCID-id: 0000-0003-3200-0374
2016 (engelsk)Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 35052Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.

sted, utgiver, år, opplag, sider
Nature Publishing Group, 2016. Vol. 6, artikkel-id 35052
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-196392DOI: 10.1038/srep35052ISI: 000385352500001PubMedID: 27748381Scopus ID: 2-s2.0-84994000069OAI: oai:DiVA.org:kth-196392DiVA, id: diva2:1050347
Merknad

QC 20161128

Tilgjengelig fra: 2016-11-28 Laget: 2016-11-14 Sist oppdatert: 2020-03-09bibliografisk kontrollert
Inngår i avhandling
1. Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
Åpne denne publikasjonen i ny fane eller vindu >>Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Fluorophore blinking dynamics are highly sensitive to the local environment and can be used as an additional readout parameter to increase the information gained from existing fluorescence techniques.The origin of these blinking patterns are photophysical transitions to and from a manifold of non-luminescent states. The long lifetime of these dark transient states, typically 103 to 106 times longer than the fluorescent state, gives them correspondingly more time to sense their environment. For this reason, fluorophore blinking dynamics are particularly sensitive to low frequency events, such as diffusion-mediated interactions between the fluorophore and dilute species.

Transient State (TRAST) monitoring has been developed to quantify fluorophore blinking dynamics in a simple and widely applicable manner. TRAST does not need to resolve individual blinking events, but instead monitors the average fluorescence intensity in response to a modulated excitation. By systematically varying the modulation parameters, the transient state kinetics of the sample are mapped out. Without the need for time-resolved detection, a regular camera can be used to image blinking dynamics with high spatial resolution.

This thesis presents TRAST characterizations of common autofluorescent compounds and demonstrates their ability to sense relevant biological parameters such as oxygen concentration and redox potential. In Papers I and II, the autofluorescent co-enzymes flavin and NAD(P)H were studied, and label-free imaging of local redox variations within cells was demonstrated. Perturbing the cells, through dilute additions of mitochondrial uncouplers, revealed a strong andlocalized response in the TRAST images. In Paper III we studied tryptophan autofluorescence and used it to detect conformational changes in an unlabeled spider silk protein.

Labeling with external fluorophores can add further specificity to the TRAST measurements. In Paper IV, TRAST was used to monitor diffusion-mediated interactions between lipids and receptors in a cell membrane, including the influence of receptor activation. In Paper V we tracked folding of RNA into G-quadruplexes in live cells, monitored via the isomerization properties of an attached cyanine dye.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2019. s. i-vi; 116
Serie
TRITA-SCI-FOU ; 2019:13
HSV kategori
Forskningsprogram
Biologisk fysik; Fysik
Identifikatorer
urn:nbn:se:kth:diva-246020 (URN)978-91-7873-142-8 (ISBN)
Disputas
2019-04-05, FB53, KTH, Roslagstullsbacken 21, Stockholm, 09:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Merknad

QC 20190312

Tilgjengelig fra: 2019-03-12 Laget: 2019-03-11 Sist oppdatert: 2019-03-13bibliografisk kontrollert

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