Change search
ReferencesLink to record
Permanent link

Direct link
On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Show others and affiliations
2010 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 18, 6197-6206 p.Article in journal (Refereed) Published
Abstract [en]

Single-molecule FRET experiments on freely diffusing rigid molecules frequently show FRET efficiency (E) distributions broader than those defined by photon statistics. It is often unclear whether the observed extra broadening can be attributed to a physical donor-acceptor distance (R-DA) distribution. Using double-stranded DNA (dsDNA) labeled with Alexa488 and Cy5 (or Alexa647) as a test system, we investigate various possible contributions to the E distribution width. On the basis of simultaneous analysis of donor and acceptor intensities and donor lifetimes, we conclude that dsDNA chain dynamics can be ruled out as a possible reason for the observed E distribution broadening. We applied a set of tools to demonstrate that complex acceptor dye photophysics can represent a major contribution to the E distribution width. Quantitative analysis of the correlation between FRET efficiency and donor fluorescence lifetime in 2D multiparameter histograms allows one to distinguish between broadening due to distinct FRET or dye species. Moreover, we derived a simple theory, which predicts that the apparent distance width due to acceptor fluorescence quantum yield variations increases linearly with physical donor-acceptor distance. This theory nicely explains the experimentally observed FRET broadening of a series of freely diffusing labeled dsDNA and dsRNA molecules. Accounting for multiple acceptor states allowed the fitting of experimental E distributions, assuming a single fixed donor-acceptor distance.

Place, publisher, year, edition, pages
2010. Vol. 114, no 18, 6197-6206 p.
National Category
Physical Chemistry
URN: urn:nbn:se:kth:diva-27903DOI: 10.1021/jp100025vISI: 000277280500027OAI: diva2:383078
QC 20110104Available from: 2011-01-04 Created: 2011-01-03 Last updated: 2012-11-26Bibliographically approved
In thesis
1. Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
Open this publication in new window or tab >>Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last fifteen years several methods have been developed for probing biomolecules (DNA, RNA, proteins) one at a time. Among these methods fluorescence spectroscopy and in particular its many implementations for monitoring Förster Resonance Energy Transfer (FRET), have attracted much interest.

This thesis deals mainly with high-precision single molecule FRET (smFRET) studies between a donor and an acceptor fluorophore attached to a biomolecule. Methodologies like multi-parameter fluorescence detection (MFD) and Probability Distribution Analysis (PDA) are used. We investigate, how and in which occasions; complex photophysical properties of the acceptor could influence the experimentally obtained FRET efficiency distributions. The value of smFRET experiments in enzymology is exemplified by presenting studies on DNA-related enzymes. Three structural conformations (Open, Closed, and Nucleotide-Binding) of Klentaq1, a DNA polymerase, have been resolved by measurements on freely diffusing molecules. We observe that the levels of occupancy of these conformations and the transitions among them, are dependent on the nature of the incoming dNTP, shedding more light into how conformational selection controls the incorporation cycle. Additionally, smFRET studies on MutS, a protein responsible for the initiation of the DNA mismatch repair machinery, have identified the existence of a preferred orientation of binding of the protein to asymmetric mismatches of DNA strands. Inhibiting MutS from binding in this preferred orientation has negative implications on the efficiency of the initiation of the overall DNA repair process.

Shifting from spectroscopy to microscopy, we use FRET imaging for monitoring interactions between the Human Epidermal Growth Receptors, HER1 and HER2, and the Insulin Growth Factor 1 Receptor, IGF1R, in fixed cells obtained from patients with suspect breast cancer lesions. While working on FRET imaging, the need for developing methodologies for the objective evaluation of the sensitivity of confocal laser scanning microscopes (CLSM) was identified. In order to provide figure of merits for the sensitivity of a microscope, we use Fluorescence Correlation Spectroscopy (FCS) and Transient State (TRAST) imaging measurements on aqueous solutions of Rhodamine 110. Our results suggest that TRAST imaging measurements could serve as a fast and easy test for the day-to-day maintenance of a CLSM and could provide reference standards for comparing images obtained by different microscope systems.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. 95 p.
Trita-FYS, ISSN 0280-316X ; 2012:87
FRET, FCS, single-molecule biophysics
National Category
urn:nbn:se:kth:diva-105748 (URN)978-91-7501-575-0 (ISBN)
Public defence
2012-12-10, FA32, AlbaNova University Center, Stockholm, 10:00 (English)

QC 20121126

Available from: 2013-04-02 Created: 2012-11-26 Last updated: 2013-04-02Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Sisamakis, Evangelos
By organisation
Experimental Biomolecular Physics
In the same journal
Journal of Physical Chemistry B
Physical Chemistry

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 66 hits
ReferencesLink to record
Permanent link

Direct link