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Structure and exchange dynamics of the apical domains of the thermosome and human saposins by NMR spectroscopy [Elektronische Ressource] / Michael John

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Department Chemie der Technischen Universitt Mnchen Structure and Exchange Dynamics of the Apical Domains of the Thermosome and Human Saposins by NMR Spectroscopy Michael John Vollstndiger Abdruck der von der Fakult t Chemie der Technischen Universitt Mnchen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. J. Buchner Prüfer der Dissertation: Univ.-Prof. Dr. H. Kessler Univ.-Prof. Dr. W. Domcke Priv.-Doz. Dr. G. Gemmecker Die Dissertation wurde am 30.06.2004 bei der Technischen Universitt Mnchen eingereicht und durch die Fakult t fr Chemie am 27.07.2004 angenommen.üü äü ä äü ä dedicated to my parents In theory, there is no difference between theory and practice. In practice, however, there is. - Unknown Abstract NMR spectroscopy provides a versatile and indispensable methodology for the characterisation of molecular structure and dynamics in modern biochemical research. A part of the present work focuses on fast structural predictions, derived from major NMR parameters and from their similarity between sequence-related proteins.

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Published 01 January 2004
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Department Chemie
der Technischen Universitt Mnchen


Structure and Exchange Dynamics
of the Apical Domains of the Thermosome
and Human Saposins
by NMR Spectroscopy


Michael John


Vollstndiger Abdruck der von der Fakult t Chemie der Technischen Universitt Mnchen
zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. J. Buchner
Prf? er der Dissertation: Univ.-Prof. Dr. H. Kessler
Univ.-Prof. Dr. W. Domcke
Priv.-Doz. Dr. G. Gemmecker

Die Dissertation wurde am 30.06.2004 bei der Technischen Universitt Mnchen eingereicht
und durch die Fakult t fr Chemie am 27.07.2004 angenommen.
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dedicated to my parents


















































In theory, there is no difference
between theory and practice.
In practice, however, there is.
- Unknown

Abstract
NMR spectroscopy provides a versatile and indispensable methodology for the
characterisation of molecular structure and dynamics in modern biochemical research. A part
of the present work focuses on fast structural predictions, derived from major NMR
parameters and from their similarity between sequence-related proteins. In two isolated apical
domains of a large archaeal chaperonin, residues whose conformation was influenced by
crystal packing interactions were shown to be highly disordered in solution. For a small class
of homologous proteins, the human saposins, residual dipolar couplings were used to validate
their common fold. Many proteins are subject to chemical and conformational exchange
processes, which are often essential for their function, but may render the molecule
unamenable for structural investigations, as in the case of the GM2 activator protein. Several
NMR-spectroscopic methods were employed to study hydrogen- and slow conformational
exchange kinetics as well as its dependence on pH and temperature in saposin D.


Zusammenfassung
Die NMR-Spektroskopie stellt der modernen biochemischen Forschung ein vielseitiges
methodisches Arsenal zur Charakterisierung von molekularer Struktur und Dynamik zur
Verfgung. Ein Teil der vorliegenden Arbeit befasst sich mit schnellen strukturellen
Vorhersagen aus wichtigen NMR-Parametern sowie deren ˜hnlichkeit in sequenziell
verwandten Proteinen. Anhand von zwei isolierten apikalen Domnen eines grossen archalen
Chaperonins konnte gezeigt werden, dass einige Aminos urereste, im Gegensatz zum Kristall,
in Lsung eine ungeordnete Konformation einnehmen. In einer kleinen Klasse von
homologen Proteinen, den humanen Saposinen, wurden dipolare Restkopplungen zur
Validierung der einheitlichen Faltung verwendet. Viele Proteine unterliegen chemischen oder
konformativen und fr ihre Funktion essentiellen Austauschprozessen, durch die sie jedoch
fr eine strukturelle Charakterisierung unzug nglich werden, wie zum Beispiel der GM2-
Aktivator. Verschiedene NMR-spektroskopische Methoden wurden eingesetzt, um Amid- und
Konformationsaustausch sowie dessen pH- und Temperaturabh ngigkeit in Saposin D zu
untersuchen.

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?Parts of this thesis have already been published:

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris,
purification, isotopic labelling and biophysical characterisation. M. Wendeler, J.
Hoernschemeyer, M. John, N. Werth, M. Schoeniger, T. Lemm, R. Hartmann, H. Kessler and
K. Sandhoff (2004) Protein Expr. Purif. 34, 147-157.

1 15 13Backbone H, N and C resonance assignments of α-ADT and β-ADT. M. John, M.
Heller, M. Coles, G. Bosch, W. Baumeister and H. Kessler (2004) J. Biomol. NMR 29, 209-
210.

NMR studies on the substrate-binding domains of the thermosome: structural plasticity
in the protrusion region. M. Heller, M. John, M. Coles, G. Bosch, W. Baumeister and H.
Kessler (2004) J. Mol. Biol. 336, 717-729.
Acknowledgement
The work presented in this thesis was done under the guidance of Prof. Dr. Horst Kessler at
the Institute for Organic Chemistry and Biochemistry of the Technical University of Munich,
Germany, in the period between July 2000 and June 2004. I would like to thank my
supervisor Prof. Dr. Horst Kessler for excellent research facilities, his confidence and the
freedom to pursue the projects with own ideas, helpful discussions and unrestricted support in
all concerns.
My further thank goes to:
● the former and present staff of the NCE Markus Heller, Melina Haupt, the kangaroo
Dr. Murray Coles, Dr. Tammo Diercks, Dr. Vinocento Vincent Truffault, Sarav
Saravanakumar Narayanan, Zhongjing Chen, Dr. Louis XIV Patard and Jochen Klages for
the great atmosphere, but also for steady scientific exchange.
● the old guys Dr. Gustav Gerd Gemmecker, Dr. Julien Furrer, Dr. Bulu Burkard Luy,
Dr. Tosh Thomas Schulte-Herbr ggen and Dr. Bernd Greif Reif for their patience being
confronted with my demand for discussions.
● the knechts Andreas Brandl, Sebastian Ahrendt, Stefanie Gasteyer, Tim Bartels, Florian
Leiss and Tobias Rossteuscher for their fresh enthusiasm.
● again Markus for carefully reading the manuscript (while preparing his own thesis !) and
helpful suggestions
● the team for spectrometers and computers Dr. Rainer Haessner, Monika Goede and Alex
Frenzel for continuously setting up the latest versions (before one is used to the old ones).
● the synthies Dr. Martin Sukopp, Armin Modlinger, Dominik Heckmann, Axel Meyer and
Timo Weide for making their facilities and knowledge available and the proof that an
interaction between chemists and spectroscopists is indeed possible.
● Mandar Deshmukh and Mrunal Pendke for an unforgettable Indian wedding, loads of
Indian food and help in preparing Xmas cookies.
● the little mermaid Cindie Kehlet for some sunny coffee breaks.
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? ?● the secretaries Beate Diaw, Marianne Machule and Evelyn
Bruckmaier for their professional work.
● the sometimes obstinate fellows Laurel, Hardy, Pat and Chef for
their company and entertainment during lonely evenings.
● all further group members for a wonderful time in the lab, on
conferences, in the mountains and at the Wies·n.
● Dr. Gundula Bosch and Prof. Dr. Wolfgang Baumeister from
the MPI for Biochemistry in Martinsried, Germany for calling the
legend ADT into being, and shipping of samples on the spot.
● Michaela Wendeler and Prof. Dr. Konrad Sandhoff from the University of Bonn for the
delivery of tons of activators and saposins.
● Prof. Dr. Ruth Gschwind, University of Bonn, for lightening the
fire of NMR spectroscopy inside me.
● the SOMA for efficient relaxation at low frequencies.
● my parents for their unlimited support.
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