| 1. |
- Cristovao, Michele, et al.
(författare)
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Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:12, s. 5448-5464
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Kalinin, Stanislav, et al.
(författare)
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On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:18, s. 6197-6206
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
- Mücksch, Jonas, et al.
(författare)
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Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains
- 2015
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Ingår i: Journal of Biophotonics. - : Wiley-VCH Verlagsgesellschaft. - 1864-063X .- 1864-0648. ; 8:5, s. 392-400
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.
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| 5. |
- Rabasovic, M. D., et al.
(författare)
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Label-Free Fluctuation Spectroscopy Based on Coherent Anti-Stokes Raman Scattering from Bulk Water Molecules
- 2016
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Ingår i: ChemPhysChem. - : John Wiley & Sons. - 1439-4235 .- 1439-7641. ; 17:7, s. 1025-1033
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticles (NPs) and molecules can be analyzed by inverse fluorescence correlation spectroscopy (iFCS) as they pass through an open detection volume, displacing fractions of the fluorescence-emitting solution in which they are dissolved. iFCS does not require the NPs or molecules to be labeled. However, fluorophores in m-mm concentrations are needed for the solution signal. Here, we instead use coherent anti-Stokes Raman scattering (CARS) from plain water molecules as the signal from the solution. By this fully label-free approach, termed inverse CARS-based correlation spectroscopy (iCARS-CS), NPs that are a few tenths of nm in diameter and at pM concentrations can be analyzed, and their absolute volumes/concentrations can be determined. Likewise, lipid vesicles can be analyzed as they diffuse/flow through the detection volume by using CARS fluctuations from the surrounding water molecules. iCARS-CS could likely offer a broadly applicable, label-free characterization technique of, for example, NPs, small lipid exosomes, or microparticles in biomolecular diagnostics and screening, and can also utilize CARS signals from biologically relevant media other than water.
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| 6. |
- Rothwell, Paul J., et al.
(författare)
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dNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase INSIGHTS INTO THE ROLE OF THE "NUCLEOTIDE-BINDING" STATE
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:19, s. 13575-13591
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Tidskriftsartikel (refereegranskat)abstract
- DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.
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| 7. |
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| 8. |
- Sisamakis, Evangelos, et al.
(författare)
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ACCURATE SINGLE-MOLECULE FRET STUDIES USING MULTIPARAMETER FLUORESCENCE DETECTION : SINGLE MOLECULE TOOLS, PT B
- 2010
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Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 474, s. 455-514
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Forskningsöversikt (refereegranskat)abstract
- In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Forster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of sm FRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape.
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| 9. |
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| 10. |
- Sisamakis, Evangelos
(författare)
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Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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