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Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0002-6191-9921
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 35052Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016. Vol. 6, 35052
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-196392DOI: 10.1038/srep35052ISI: 000385352500001ScopusID: 2-s2.0-84994000069OAI: oai:DiVA.org:kth-196392DiVA: diva2:1050347
Note

QC 20161128

Available from: 2016-11-28 Created: 2016-11-14 Last updated: 2016-11-28Bibliographically approved

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Hevekerl, HeikeTornmalm, JohanWidengren, Jerker
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CiteExportLink to record
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