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Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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.
Series
Trita-FYS, ISSN 0280-316X ; 2012:87
Keyword [en]
FRET, FCS, single-molecule biophysics
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-105748ISBN: 978-91-7501-575-0 (print)OAI: oai:DiVA.org:kth-105748DiVA: diva2:571945
Public defence
2012-12-10, FA32, AlbaNova University Center, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20121126

Available from: 2013-04-02 Created: 2012-11-26 Last updated: 2013-04-02Bibliographically approved
List of papers
1. On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits
Open this publication in new window or tab >>On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits
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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.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-27903 (URN)10.1021/jp100025v (DOI)000277280500027 ()
Note
QC 20110104Available from: 2011-01-04 Created: 2011-01-03 Last updated: 2017-12-11Bibliographically approved
2. dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1: insights into the role of the "nucleotide binding" state
Open this publication in new window or tab >>dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1: insights into the role of the "nucleotide binding" state
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(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-105745 (URN)
Note

QS 2012

Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2012-11-26Bibliographically approved
3. Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA
Open this publication in new window or tab >>Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 12, 5448-5464 p.Article in journal (Refereed) Published
Abstract [en]

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.

Keyword
Resonance Energy-Transfer, Repair Protein Muts, Probability-Distribution Analysis, Recognition Complex, Tetramerization Domain, Atpase Activity, Sliding Clamp, Hydrolysis, Msh2-Msh6, Fret
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-99403 (URN)10.1093/nar/gks138 (DOI)000305829000030 ()2-s2.0-84863204327 (Scopus ID)
Funder
EU, European Research Council, MOBILITY-1 19566 HEALTH-F4-2008-223545
Note
QC 20120730Available from: 2012-07-30 Created: 2012-07-30 Last updated: 2017-12-07Bibliographically approved
4. Assessment of the sensitivity of a confocal laser scanning microscope by fluorescence correlation spectroscopy and TRAST imaging
Open this publication in new window or tab >>Assessment of the sensitivity of a confocal laser scanning microscope by fluorescence correlation spectroscopy and TRAST imaging
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(English)Manuscript (preprint) (Other academic)
National Category
Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:kth:diva-105741 (URN)
Note

QS 2012

Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2013-01-07Bibliographically approved

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Citation style
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Output format
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