Fluorescence resonance energy transfer between multiple chromophores studied by single-molecule spectroscopy [Elektronische Ressource] / Alessandro Valeri

Alessandro Rubini

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2010
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Fluorescence Resonance Energy Transfer
between multiple chromophores studied by
single-molecule spectroscopy


Thesis


Department of Mathematics and Natural Sciences
Heinrich-Heine University Düsseldorf





Alessandro Valeri
from Rome
February 2009
Institute of Molecular Physical Chemistry,
Heirich-Heine University Düsseldorf









Printed with the permission of
Department of Mathematics and Natural Sciences
Heinrich-Heine-University Düsseldorf




1. Referee: Prof. Dr. Claus A.M. Seidel
2. Referee: Prof. Dr. Filipp Oesterhelt

Date of the oral examination: 06.05.2009
This thesis is based on the following papers:

Filtered FCS and Species Cross Correlation Function.
Felekyan, S., Kalinin, S., Valeri, A., Seidel, C. A. M, Proceedings of SPIE, 2009, accepted.

Characterizing Multiple Molecular States in Single-molecule Multi-parameter
Fluorescence Detection by Probability Distribution Analysis.
Kalinin, S., Felekyan, S., Valeri, A., Seidel, C. A. M, Journal of Physical Chemistry B, 2008,
112 (28), p. 8361-8374.

Probability Distribution Analysis of a Two-State Interconverting FRET System.
Kalinin, S., Valeri, A., Antonik, M., Felekyan, S., Seidel, C. A. M, manuscript.

Nucleosome disassembly intermediates characterized by single-molecule FRET.
Gansen, A., Valeri, A., Hauger, F., Felekyan, S., Kalinin, S., Tóth, K., Langowski, J., Seidel,
C.A.M., manuscript.


Posters at international conferences:
Molecular Ångström Optics: A dynamical view of biomolecular structure.
Seidel, C. A. M., Felekyan, S., Kalinin, S., Oesterhelt, F., Valeri, A., Wo źniak, A. K.
Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress, Long
Beach, February 02-06, 2008, Long Beach, USA.

Dynamics of individual nucleosomes analysed with single molecule.
Gansen, A., Hauger, F., Toth, K., Valeri, A., Felakyan, S., Seidel, C. A. M., Langowski, J.
Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress, Long
Beach, February 02-06, 2008, Long Beach, USA.

Multiparameter Fluorescence Detection, theory and application to Two-Step FRET.
Valeri, A., Tojira, O., Felekyan, S., Kudryavtsev, V., Antonik, M., Muller, P.A., Seidel, C. A.
M.
9th Conference on Methods and Applications of Fluorescence (MAF-9), September 04 – 07,
2005, Lisbon, Portugal.

Conformational dynamics of special nucleic acid structures studied by single molecule FRET.
AlessandroValeri, Enno Schweinberger, Volodymyr Kudryavtsev, Marcelle König, Pierre-
Alain Muller, Rüdiger Dede, Suren Felekyan, Claus A. M. Seidel
th9 International Workshop on "Single Molecule Detection and Ultra Sensitive Analysis in the
Life Sciences" September 24-26, 2003, Berlin, Germany.

Conformational Dynamics of special Nucleic Acid Structures Studied by Single Molecule
FRET.
E. Schweinberger, A. Valeri, M. König, V. Kudryavtsev, P. A. Muller, R. Dede, S. Felekyan,
C. A. M. Seidel
8th Conference on Methods and Applications of Fluorescence (MAF-8), August 24-27, 2003,
Prague, Czech Republic.
CONTENTS
1 INTRODUCTION 1
2 FLUORESCENCE 5
2.1 Fluorescence anisotropy 7
2.2 Fluorescence Resonance Energy Transfer (FRET) 8
3 INSTRUMENTATION 13
3.1 UV-VIS spectrophotometer 13
3.2 Steady state fluorometers 13
3.3 Time Correlated Single Photon Counting (TCSPC) 14
3.4 Confocal microscope setup 14
3.4.1 Fluorescence signals 16
3.4.2 Anisotropy 18
3.4.3 Colours 18
4 FLUORESCENCE CORRELATION SPECTROSCOPY (FCS) 19
4.1 Filtered Fluorescence Correlation Spectroscopy (fFCS) 21
4.1.1 Fluorescence Lifetime Filters (FLF) 22
5 MULTI-PARAMETER FLUORESCENCE DETECTION (MFD) 25
5.1 Two-dimensional plots 25
5.2 MFD equations 26
5.2.1 Dye quenching 27
5.2.2 Interconversion of FRET states 29
5.2.3 Dye linker movement 32
5.3 Conversion of fluorescence intensity ratio into E and R 34 FRET DA
6 PROBABILITY DISTRIBUTION ANALYSIS (PDA) 37
6.1 Theory 37
6.1.1 FRET experiments 40
6.1.2 Polarisation experiments 41
6.2 Multiple species and brightness correction 42
6.3 Multi-molecular events 43
6.4 Dynamic systems 43
6.5 Dye heterogeneities 44
7 TWO-STEP FRET 47
7.1 Material and methods 47
7.2 Single-molecule experiments 49
7.2.1 Donor only molecules 50
7.2.2 DA2 molecules 52
7.2.3 A1 containing molecules 54
7.3 Distances 57
7.4 Conclusions 57
8 FOUR-WAY DNA JUNCTIONS 59
8.1 Magnesium dependence 60
8.2 Material and methods 61
8.3 Single-molecule experiments 64
8.4 Dynamic PDA 67
8.5 Alternative kinetic models 72
8.6 FCS 73
8.7 Comparison between PDA, FCS and other techniques 77
8.8 Comparison with other Holliday Junction sequences 85
8.9 Conclusions 85
9 NUCLEOSOMES 87
9.1 Single-molecule experiments 87
9.2 Geometrical model 88
SUMMARY 91
LITERATURE 94

1 Introduction
In all biological processes that rely on molecular interactions there is a precise link between
structure and function. Biomolecules, however, do not have static structures, they rather
fluctuate between conformations and their functions “are governed ultimately by their
dynamic character (or personality)” (Henzler-Wildman and Kern 2007). It has been shown,
for example, that it is the dynamic nature of an enzyme that characterises its activity
(Eisenmesser, Millet et al. 2005), and that in a broad class of proteins, folding takes place
only upon binding to a substrate, intrinsically disordered proteins (Dyson and Wright 2005).
Thus, to understand those processes that are at the base of life, one has to study not only the
static molecular structures but also the conformational changes each molecule undergoes prior
and during interaction with its partners.
Since the first observations of single molecules with optical detection methods (Hirschfeld
1976; Moerner and Kador 1989) fluorescence spectroscopy has become an important tool in
the study of the dynamic and conformational properties of biomolecules (Moerner and Fromm
2003). In single-molecule experiments, in fact, ensemble averaging is avoided and the
information on the heterogeneities and dynamic properties of a system is directly accessible
(Kühnemuth and Seidel 2001).
Fluorescence emission is highly sensitive to the chromophore’s environment therefore
conformational changes in the immediate surrounding of the fluorescent probe can be easily
detected. However, when molecular interactions induce long-range conformational changes,
one chromophore may not be enough to obtain detailed information. This problem is
overcome by labelling the molecule (or the molecular assembly) with multiple dyes so that
Fluorescence Resonance Energy Transfer (FRET) (Förster 1948) can occur. In FRET, energy
is transferred non-radiatively from an excited donor to an acceptor chromophore. The
efficiency of the transfer is strongly dependent on the distance and mutual orientation of the
dyes, thus intra- and inter-molecular distances, and their fluctuations, can be assessed with
high accuracy. The distances that can be probed are comparable to the size of the molecules,
10-100Å, and for this reason FRET has been used as a molecular ruler (Stryer and Haugland
1967).
A chromophore possesses multiple fluorescence dimensions (intrinsic properties): the spectral
properties of absorption and emission, fluorescence brightness and quantum yield, Φ , F
fluorescence lifetime, τ, and fluorescence anisotropy, r. With the use of multiple
chromophores even more dimensions are available (intrinsic properties of donor and acceptor
1and donor-acceptor distances via FRET). Multi-parameter fluorescence detection (MFD)
(Eggeling, Berger et al. 2001) is a single-molecule technique that allows one to record
simultaneously all these fluorescence parameters and thus, give access to a wealth of
information which can be used to successfully investigate and characterise complex systems.
Moreover MFD is a time-resolved technique, making the time evolution of the system another
available parameter.
In this work it is shown how to combine MFD with other techniques, Fluorescence
Correlation Spectroscopy and Probability Distribution Analysis, to enhance their capabilities.

Fluorescence Correlation Spectroscopy.
Fluorescence Correlation Spectroscopy (FCS) is a powerful technique used to study all those
processes that induce a fluctuation of the fluorescence signal (Magde, Elson et al. 1972;
Magde, Elson et al. 1974). The characteristic relaxation times that describe the kinetic
properties of each process are obtained in FCS by fitting the correlation function of the
fluorescence signal. A wide range of processes, spanning over several order of correlation
times, are accessible to FCS: translational and rotational diffusion (Mets and Rigler 1994),
chemical reactions (Elson and Magde 1974; Palmer III and Thompson 1987) and
conformational changes (Widengren and Mets 2002). The limits in single-molecule FCS are