Analysis of large-scale structural changes in proteins with focus on the recovery stroke mechanism of myosin II [Elektronische Ressource] / vorgelegt von Sidonia E. Mesentean

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INAUGURAL – DISERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg Vorgelegt von Dipl.-Ing. Sidonia E. Mesentean aus Musatesti/Arges, Romania Tag der mündlichen Prüfung: 20.07.2007 Analysis of Large-Scale Structural Changes in Proteins with focus on the Recovery Stroke Mechanism of Myosin II Gutachter: Prof. Dr. Joachim Spatz Prof. Dr. Jeremy C. Smith Summary The mechanisms through which proteins achieve their functional three-dimensional structure starting from a string of amino acids, as well as the manner in which the interactions between different structural elements are orchestrated to mediate function are largely unknown, despite the large amount of data accumulating from theoretical and experimental studies. One clear view emerging from all these studies is that function is a result of the intrinsic protein dynamics and flexibility, namely the motions of its well-defined structural elements and their ability to change their position and shape in space to allow large conformational transitions necessary for the function.

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INAUGURAL – DISERTATION


zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen
Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg






Vorgelegt von
Dipl.-Ing. Sidonia E. Mesentean
aus Musatesti/Arges, Romania


Tag der mündlichen Prüfung: 20.07.2007



















































Analysis of Large-Scale Structural Changes
in Proteins with focus on the Recovery
Stroke Mechanism of Myosin II




















Gutachter: Prof. Dr. Joachim Spatz
Prof. Dr. Jeremy C. Smith
































Summary


The mechanisms through which proteins achieve their functional three-
dimensional structure starting from a string of amino acids, as well as the manner in
which the interactions between different structural elements are orchestrated to
mediate function are largely unknown, despite the large amount of data accumulating
from theoretical and experimental studies. One clear view emerging from all these
studies is that function is a result of the intrinsic protein dynamics and flexibility,
namely the motions of its well-defined structural elements and their ability to change
their position and shape in space to allow large conformational transitions necessary
for the function.
Simulation techniques have been increasingly used over the past years in the
endeavour to solve the structure-function puzzle as they have proven to be powerful
tools to investigate the dynamics of proteins. However, extracting useful dynamical
information from trajectories thus generated in order to draw functionally relevant
conclusions is not always straight forward, especially when the protein function
involves concerted movements of entire protein domains. This is due to the high
dimensionality of the energy surface the proteins can explore. Therefore, a decrease in
complexity is to be desired and can be achieved in principle by reducing the number of
dimensions to the ones capturing only the dominant motions of the protein.
To this purpose, in this thesis two different dimensionality reducing
techniques, namely Principal Component Analysis and Sammon Mapping are applied
and compared on four proteins that undergo conformational changes with different
amplitudes and mechanisms. In particular, the present thesis tackles the large
conformational change occurring during the recovery stroke of myosin, using these
methods and rigidity analysis algorithms in the attempt to elucidate in atomic detail the
structural mechanism underlying the function of this protein that couples ATP
hydrolysis to the mechanical force needed to achieve muscle contraction.
The results presented in this thesis show the successful applicability of certain
dimensionality reducing methods to large conformational changes and their suitability
in analyzing and dissecting dynamical transitions in computationally generated
trajectories. The findings regarding the recovery stroke step in the myosin cycle are
consistent with experimental data coming from mutational studies and confirm the previously postulated communication mechanism between the active sites of the
protein, thus representing a major contribution to the field of molecular motors and a
strong evidence of the importance of theoretical studies in complementing the
experimental investigations.































Zusammenfassung


Der Mechanismus durch welchen Proteine, ausgehend von einer Folge von
Aminosäuren, ihre funktionsfähige dreidimensionale Struktur erlangen, sowie auch die
Art und Weise, in der die Wechselwirkungen zwischen verschiedenen
Strukturelementen orchestriert sind, um Funktion zu vermitteln, ist, trotz der großen
Datenmengen, die sich aus theoretischen und experimentellen Studien angesammelt
haben, größtenteils unbekannt. Eine Anschauung, die sich aus all diesen
Untersuchungen herausbildet, ist, dass Funktion ein Resultat der intrinsischen
Proteindynamik und -flexibilität ist, und zwar der Bewegungen ihrer wohldefinierten
Strukturelemente und deren Fähigkeit, ihre Position und Form zu verändern, um große,
für die Funktion notwendige, Konformationsänderungen zu ermöglichen.
In den letzten Jahren sind vermehrt Simulationstechniken in dem Bestreben
eingesetzt worden, das Struktur-Funktions-Puzzle zu lösen, da sie sich als mächtige
Werkzeuge zur Erforschung der Dynamik von Proteinen erwiesen haben. Nützliche
dynamische Informationen aus den so erzeugten Trajektorien zu extrahieren, um
daraus funktionsrelevante Schlüsse zu ziehen, ist allerdings nicht immer einfach,
besonders wenn die Arbeitsweise des Proteins mit gemeinschaftlichen Bewegungen
ganzer Domänen einhergeht. Dies liegt an der hohen Dimensionalität der
Energiefläche, die Proteine ablaufen können. Daher ist eine Verringerung der
Komplexität erwünscht und kann im Prinzip durch Reduktion der Dimensionen auf
jene, welche die dominanten Bewegungen des Proteins erfassen, erreicht werden.
Zu diesem Zweck werden in dieser Arbeit zwei dimensionsreduzierende
Techniken, nämlich Hauptkomponentenanalyse (principal component analysis) und
Sammon Abbildung (Sammon mapping) auf vier Proteine angewendet und verglichen,
die Konformationsänderungen verschiedenen Umfangs und verschiedener
Mechanismen durchlaufen. Vornehmlich befasst sich die vorliegende Arbeit mit der
großen Konformationsänderung während des “recovery stroke” von Myosin. Die
genannten Methoden werden zusammen mit Rigiditätsanalysen benutzt, um den
strukturellen Mechanismus aufzuklären, welcher der Funktion dieses Proteins, das
ATP-Hydrolyse an die mechanische Kraft koppelt, welche zur Muskelkontraktion
benötigt wird, zugrunde liegt. Die in dieser Arbeit dargestellten Ergebnisse zeigen die erfolgreiche
Anwendbarkeit bestimmter dimensionsreduzierender Methoden auf große
Konformationsänderungen und deren Eignung für die Analyse und Aufgliederung
dynamischer Übergänge in computergenerierten Trajektorien. Die Erkenntnisse
hinsichtlich des “recovery strokes” im Myosin-Zyklus sind im Einklang mit
experimentellen Daten aus Mutationsstudien und bestätigen den zuvor postulierten
Kommunikationsmechanismus zwischen den aktiven Zentren des Proteins, und stellen
daher einen bedeutenden Beitrag auf dem Gebiet der molekularen Motoren und einen
deutlichen Beweis für die Wichtigkeit theoretischer Studien in Ergänzung zu
experimentellen Untersuchungen dar.


























Acknowledgments



The content of these acknowledgment pages are at least as important for me as the
obtained results presented in the following pages. Without the persons mentioned here
none of the published work would have been possible.
First of all, I want to thank to Prof. Jeremy C. Smith for giving me the chance to work
with him. His guidance along my PhD work was like a road, always showing the
direction along which I have to go in order to achieve my purpose. His charm and
geniality was present not only during the working hours but also in the social life of his
group. Another mentor to which I want to thank is Dr. Stefan Fischer. He always knew
how to bring into the light the best part of my work. He was walking with me side by
side through the problems arising from the tasks I had to complete.
To my husband I am deeply indebted for his permanent support, for his unique way of
bringing me back on the track when I was lost, for listening my complains and worries
and not the last for his love. Together with my family, they found the perfect words for
cheering me up and the best mood for celebrating.
A special thank to my parents for their love and for believing in me till the end. Also
for fighting against the social and economical hindrances present along my way, they
deserve a special place in my mind and heart. My sister also played an important role
due to the fact that she was my shadow in all this years and followed my spirit all
through the way.
I miss the words when I have to express my gratitude for the help received from dear
friends like Crina, Andreea, Durba or Bogdan. They always surrounded me with hope
and optimism, independent if it was work, celebrations or just simple discussions.
I would like to thank Ms. Ellen Vogel for her kindness and patience with which she
solved most of my administrative matters and for always finding a few minutes to hear
my complains. Lots of thanks to all the members of the Computational Molecular
Biophysics and Computation Biochemistry groups, for helpful input and discussions
regarding my research but also for making my stay in Heidelberg unforgettable.
Last but not least I would like to acknowledge the Bundesministerium für Bildung und
Forschung and the Deutsche Forschungsgemeinschaft which financed most of this
project.