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NMR spectroscopic investigations reveal insights into antibody secretion, amyloidogenic folding intermediates, and the process of focal adhesion [Elektronische Ressource] / Sandra Groscurth

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NMR spectroscopic investigations revealinsights into antibody secretion,amyloidogenic folding intermediates,and the process of focal adhesionSandra GroscurthDissertation 2009¨ ¨TECHNISCHE UNIVERSITAT MUNCHEN¨ ¨TECHNISCHE UNIVERSITAT MUNCHENDepartment ChemieLehrstuhl IIfu¨r Organische ChemieNMR spectroscopic investigations revealinsights into antibody secretion,amyloidogenic folding intermediates,and the process of focal adhesionSandra GroscurthVollst¨andiger Abdruck der von der Fakult¨at fu¨r Chemie der Technischen Universit¨atMu¨nchen zur Erlangung des akademischen Grades einesDoktors der Naturwissenschaftengenehmigten Dissertation.Vorsitzender: Univ.-Prof. Dr. M. SattlerPru¨fer der Dissertation: 1. Univ.-Prof. Dr. H. Kessler2. Univ.-Prof. Dr. Th. Kiefhaber3. Univ.-Prof. Dr. J. BuchnerDie Dissertation wurde am 21.01.2009 bei der Technischen Universit¨at Mu¨ncheneingereicht und durch die Fakult¨at fu¨r Chemie am 03.03.2009 angenommen.Those who are not shockedwhen they first come acrossquantum mechanics cannotpossibly have understood it(Niels Henrik David Bohr)AcknowledgementThe work presented in this thesis was prepared from May 2005 until October 2008 inthe group of Prof. Dr. Horst Kessler at the Department of Chemistry of the TechnicalUniversity of Munich, Germany.I would like to thank my supervisor Prof. Dr.

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Published 01 January 2009
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NMR spectroscopic investigations reveal
insights into antibody secretion,
amyloidogenic folding intermediates,
and the process of focal adhesion
Sandra Groscurth
Dissertation 2009
¨ ¨TECHNISCHE UNIVERSITAT MUNCHEN¨ ¨TECHNISCHE UNIVERSITAT MUNCHEN
Department Chemie
Lehrstuhl II
fu¨r Organische Chemie
NMR spectroscopic investigations reveal
insights into antibody secretion,
amyloidogenic folding intermediates,
and the process of focal adhesion
Sandra Groscurth
Vollst¨andiger Abdruck der von der Fakult¨at fu¨r Chemie der Technischen Universit¨at
Mu¨nchen zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. M. Sattler
Pru¨fer der Dissertation: 1. Univ.-Prof. Dr. H. Kessler
2. Univ.-Prof. Dr. Th. Kiefhaber
3. Univ.-Prof. Dr. J. Buchner
Die Dissertation wurde am 21.01.2009 bei der Technischen Universit¨at Mu¨nchen
eingereicht und durch die Fakult¨at fu¨r Chemie am 03.03.2009 angenommen.Those who are not shocked
when they first come across
quantum mechanics cannot
possibly have understood it
(Niels Henrik David Bohr)Acknowledgement
The work presented in this thesis was prepared from May 2005 until October 2008 in
the group of Prof. Dr. Horst Kessler at the Department of Chemistry of the Technical
University of Munich, Germany.
I would like to thank my supervisor Prof. Dr. Kessler for giving me the opportunity to
join his group, for the excellent research facilities, for the unrestricted support, and for
giving me lots of personal freedom.
My further thank goes to:
• MyproteinfactoryMatthiasFeigeandMoritzMarcinowski. Thankstothemand
Prof. Dr. Johannes Buchner for the great cooperation in the antibody projects.
Furthermore, I would like to thank Matthias and Moritz for the excellent team
work and a lot of fun besides the science. Thanks guys for the wonderful time!
• Dr. Jochen Klages for his kind to elate me for the NMR spectroscopy, for an-
swering nearly every question, for the conversations about all the world and his
brother during the morning coffee, and for discovering the advantage of a banana
box.
• Prof. Dr. Thomas James and Dr. Mark Kelly, University of California, San Fran-
cisco, USA, for giving me the opportunity to join the research group for half a
year. AdditionallyProf.Dr.DianeBarberandDr.Jyoti Srivastavaforallthe cell
biological background and experiments on talin. And special thanks to Mark for
involving me in the talin project and giving me a lot of freedom in every respect,
turning my stay in San Francisco into a memorable time.
• Prof. Dr. Linda Hendershot and Dr. Yuichiro Shimizu, St. Jude Children’s Re-
search Hospital, Memphis, USA, forthe in vivo experiments onthe IgGantibody
molecules.
• Dr. Emanuele Paci and Zu Thur Yew, University of Leeds, United Kingdom, for
performing the MD simulations for the folding intermediate.
• Prof. Dr. Hermann Bauer, Georg Simon Ohm University of Applied Science,
Nuremberg, for the enjoyable tea breaks at his office and for putting in a good
word for me with Prof. Dr. Kessler.• Dr.VincentTruffaultandDr.MurrayColesforinstructingmeinsettingupNMR
experiments, for providing helpful scripts to facilitate the analysis of NMR data,
and for their endless support regarding any NMR question.
• Sylvain Tourel for the perfect team work in the biochemistry lab and for his
assistance in getting NMR amounts of pure and soluble protein.
• Dr. Axel Meyer, Dr. Jochen Klages, and Matthias Feige for critical reading of my
thesis and providing constructive suggestions.
• Dr. Rainer Haessner for his efforts to keep the spectrometers running and thus
enabling the excellent research facilities.
Finally, I would like to thank my parents for their unlimited support at all times.
And last but not least I thank Axel for pushing me just a bit to finish writing, for
always caring about exceptional alternations from science, and for all these wonderful
memories of this time in Munich.Parts of this thesis have been published:
M. Feige, S. Groscurth, M. Marcinowski, Z. Yew, V. Truffault, E. Paci, H. Kessler, J.
Buchner, ’The structure of a folding intermediate provides insight into differences in
immunoglobulin amyloidogenicity’, Proc. Natl. Acad. Sci. USA 2008, 105, 13373 –
13378.
J. Srivastava, G. Barreiro, S. Groscurth, A. Gingras, B. Goult, D. Critchley, M. Kelly,
M.Jacobson,D.Barber,’StructuralmodelandfunctionalsignificanceofpH-dependent
talin-actin binding for focal adhesion remodeling’, Proc. Natl. Acad. Sci. USA 2008,
105, 14436 – 14441.
M. Feige, S. Groscurth, M. Marcinowski, Y. Shimizu, L. Hendershot, H. Kessler, J.
Buchner, ’A natively unfolded domaincontrols thesecretion ofmurine IgGantibodies’,
submittedContents
1 Introduction 1
2 Theoretical Aspects 5
2.1 Protein Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.1 Dynamics in the ps to ns Time Scale . . . . . . . . . . . . . . . 8
2.1.2 Dynamics in the μs to ms Time Scale . . . . . . . . . . . . . . . 13
2.2 Amide Proton Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3 Residual Dipolar Couplings . . . . . . . . . . . . . . . . . . . . . . . . 18
3 The C Domain Explains Differences in Ig Amyloidogenicity 23L
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1 The Antibody Molecule Immunoglobulin G . . . . . . . . . . . . 24
3.1.2 The Immunoglobulin Fold . . . . . . . . . . . . . . . . . . . . . 29
3.1.3 Immunoglobulin Amyloidogenicity . . . . . . . . . . . . . . . . . 32
3.2 Folding Pathway of the C Domain . . . . . . . . . . . . . . . . . . . . 34L
3.2.1 Backbone Assignment . . . . . . . . . . . . . . . . . . . . . . . 35
3.2.2 Secondary Structure . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2.3 Folding Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Structural Characterization of the Trapped Folding Intermediate . . . . 42
3.3.1 Backbone and Aliphatic Side Chain Assignment . . . . . . . . . 42
3.3.2 Comparison of Secondary and Tertiary Structure of the Wild
Type and the Mutant . . . . . . . . . . . . . . . . . . . . . . . . 44
3.3.3 NMR-restrained MD Simulations . . . . . . . . . . . . . . . . . 47
3.4 Amyloidogenic Properties of the C Domain and β -microglobulin . . . 49L 2
3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4 The C 1 Domain Controls the Secretion of IgG Molecules 53H
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.1.1 Secretion of Antibody Molecules . . . . . . . . . . . . . . . . . . 54
4.1.2 Intrinsically Disordered Proteins . . . . . . . . . . . . . . . . . . 57
4.2 Investigation of the Structural Characteristics of the C 1 Domain . . . 62H
4.2.1 The Unassembled C 1 Domain is Natively Unfolded . . . . . . 62H
4.2.2 Association coupled Folding of the C 1 Domain . . . . . . . . . 65H
4.3 Insights into the Secretion Control Mechanism . . . . . . . . . . . . . . 69
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
vii