Towards the understanding of fluorescence quenching mechanisms [Elektronische Ressource] : molecular dynamics simulations of dye-quencher interactions in biomolecular systems / presented by Andrea C. Vaiana

Philip Tinnefeld

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2004
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation
submitted to the
Combined Faculties
for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences











Presented by
Dipl.-Phys. Andrea C. Vaiana
Born in Cambridge MA (USA)
Oral examination: 02.12.2004




Towards the Understanding of Fluorescence Quenching
Mechanisms: Molecular Dynamics Simulations of Dye-Quencher
Interactions in Biomolecular Systems.














Referees: Prof. Dr. Jeremy C. Smith
Prof. Dr. Heinz Horner Zusammenfassung
Der Nachweis von p53-Antikörpern ist ein wichtiges Diagnoseverfahren zur
Krebsfrüherkennung und Verlaufskontrolle. Mit Hilfe des Farbstoffes MR121 kann ein
solcher Nachweis mittels Fluoreszenspektroskopie geführt werden. Grundlage der
Fluoreszenzlöschung (quenching) des Farbstoffes ist dessen bimolekulare Wechselwirkung
mit Tryptophanresten. Die molekularen Mechanismen, die dieser Fluoreszenzlöschung zu
Grunde liegen, sind weitestgehend unbekannt. In der vorliegenden Arbeit werden MD-
Kraftfeldparameter für die Farbstoffe Rhodamine 6G (R6G) und MR121 entwickelt und die
Wechselwirkungen zwischen Farbstoff und Quencher (Trp) mittels Molekulardynamik
Simulationen untersucht. Zur Bereitstellung eines CHARMM Kraftfelds für beide oben
genannten Farbstoffe wurde eine neue, automatisierte Optimisierungsmethode für
Kraftfeldparameter von Molekülen kleinerer und mittlerer Größe entwickelt. Als
Referenzdaten dienten ab initio quanten-chemische Berechnungen. In einem zweiten Schritt
wurden MD-Simulationen zweier Farbstoff/Quencher-Systeme durchgeführt (R6G/Trp und
MR121/Trp). Die Untersuchung dieser Modellsysteme ergab wichtige Einsichten in
bimolekulare Wechselwirkungen zwischen Farbstoff und Quencher. Basierend auf Abstand
und relativer Orientierung des Systems, konnte eine quantitative Interpretation des
Quenching-Mechanismus abgeleitet werden. Die an diesen Modellsystemen gewonnenen
Resultate dienten dann zur Interpretation der Simulationsdaten zweier, am N-Terminus mit
obigen Farbstoffen markierten, Epitope des Tumorsupressor Proteins p53. In allen Phasen der
Doktorarbeit wurde gezielt versucht, die theoretischen Resultate mit verfügbaren
experimentellen Daten zu vergleichen oder zu kombinieren.

Abstract
The presence of antibodies directed against p53 in human blood serum is a specific and
independent marker of cancer. The present thesis is a simulative study of two fluorescent dyes
(rhodamine 6G and MR121) used in the development of fluorescence-based immunoassays
for the detection of p53 antibodies. The "selective" fluorescence quenching property of
tryptophan residues present in dye-conjugated peptide chains enables monitoring of
conformational dynamics and antibody binding events by means of fluorescence
spectroscopy. The molecular mechanisms of the quenching of fluorescent dyes are mostly
unknown. Here MD simulations are used in combination with existing results from ensemble
fluorescence experiments in order to obtain predictive theoretical insight into dye/quencher
interactions. A new automated refinement method was developed for deriving reliable
molecular mechanics force field parameters for small- to medium-sized molecules using
reference data from high level ab initio quantum chemical calculations. Using this method
CHARMM force field parameters for the two dyes were derived. The parameters were then
used to perform MD simulations on two simplified, but realistic dye quencher systems:
MR121/TRP and R6G/TRP. Results of these simulations have given important insight on the
bimolecular interactions between the dyes and the quencher. A quantitative interpretation of
the quenching mechanism based on it’s dependence on dye/quencher distance and orientation
has emerged. These results were then applied to the interpretation of simulation data of an
epitope from the tumor suppressor protein p53 which was labelled at the N-terminus first with
MR121 and then with R6G. In all phases of the thesis care was taken to confront and/or
combine the theoretical results with available experimental data.
"..., so solid from a distance, is riven into sects and clans,
which radiate and join, and change their names according to
the aspect from which they are approached. Study it for years
with the best teachers, and when you raise your head nothing
they have told you quite fits."

E.M. Forster on Hinduism
"A Passage to India"
Table of Contents i
TABLE OF CONTENTS
1. INTRODUCTION ....................................................................................................1
1.1. MOLECULAR MODELLING AND SIMULATION .....................................................2
1.3. MDYNAMICS SIMULATION OF BIOMOLECULES ...............................3
REFERENCES ........................................................................................................8


2. THEORY AND METHODS ...............................................................................11
2.1. QUANTUM CHEMICAL CALCULATIONS..............................................................11
2.1.1. Hartree-Fock............................................................11
2.2. MOLECULAR DYNAMICS SIMULATIONS.............................................................14
2.2.1. Force Fields - the Empirical Potential Energy Function.........................17
2.2.2. Energy Minimization...............................................................................26
2.2.3. Normal Mode Analysis...........28
2.2.4. Molecular Dynamics Simulations...........................................................30
2.3. FLUORESCENCE...................................................................32
2.3.1. Absorption and Emission of Light by Molecules...32
2.3.2. Fluorescence and Quenching of Dyes ....................................................33
REFERENCES ......................................................................................................40

3. MOLECULAR MECHANICS FORCE FIELD PARAMETERI-
SATION OF R6G AND MR121..................................................................................43 Table of Contents ii
3.1. AUTOMATED FREQUENCY MATCHING METHOD FOR FORCE FIELD
PARAMETERISATION...................................................................................................43
3.1.1. MM Force Field Parameterisation...........................43
3.1.2. AFMM and the CHARMM Force Field .................................................45
3.1.3. Calculations ............................................................50
3.2. PARAMETERISATION OF R6G AND MR121 .......................................................52
3.2.1. R6G Parameters................................52
3.2.2. R6G Crystal Structure.............................................55
3.2.3. R6G MR121 Parameters ........................................57
3.3. CONCLUSIONS .....................................................................59

REFERENCES ......................................60

4. MD SIMULATION OF TWO SIMPLE BUT REALISTIC
DYE/QUENCHER SYSTEMS: R6G/TRP AND MR121/TRP IN
EXPLICIT WATER........................................................................................................63
4.1. RESULTS OF FLUORESCENCE QUENCHING EXPERIMENTS...............................63
4.2. MD SIMULATIONS ..............................................................................................66
4.2.1. Simulation Protocol.................66
4.2.2. Potential of Mean Force: Theoretical Background .................................67
4.2.3. Simulation Results...................................................................................69
4.3. CONCLUSIONS .....................................................................................................77

REFERENCES ......78

5. MD SIMULATIONS OF A FLUORESCENCE LABELLED p53
EPITOPE.............................................................................................................................79