Structural and functional analysis of RIG-I like helicases [Elektronische Ressource] : modulating spectral properties of the green fluorescent protein with nanobodies / Axel Kirchhofer

Axel Kirchhofer

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München


Structural and Functional Analysis
of RIG-I Like Helicases

Modulating Spectral Properties of the
Green Fluorescent Protein with Nanobodies


Axel Kirchhofer

aus

Mönchengladbach

München, 2009 Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Dr. Karl-Peter Hopfner betreut.

Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfsmittel erarbeitet.

München, am 30.09.2009


...........................................
(Axel Kirchhofer)




Dissertation eingereicht am: 12.10.2009
1. Gutachter: Herr Prof. Dr. Karl-Peter Hopfner
2. Gutachter: Herr Prof. Dr. Roland Beckmann
Mündliche Prüfung am: 25.11.2009





This thesis has been prepared from June 2006 to September 2009 in the laboratory of
Professor Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-University of
Munich (LMU).

Parts of this thesis have been published in the following scientific journals:
Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, Pellis M, Muyldermans S,
Delucchi CC, Cardoso MC, Leonhardt H, Hopfner KP and Rothbauer U (2009). “Modulating
conformation and spectral properties of fluorescent proteins with nanobodies in living cells.”
Manuscript accepted at Nat Struct Mol Biol.

Pippig DA, Hellmuth JC, Cui S, Kirchhofer A, Lammens K, Lammens A, Schmidt A,
Rothenfusser S, Hopfner KP (2009). “The regulatory domain of the RIG-I family ATPase
LGP2 senses double-stranded RNA.” Nucleic Acids Res. 37(6):2014-25

Myong S, Cui S, Cornish PV, Kirchhofer A, Gack MU, Jung JU, Hopfner KP, Ha T (2009).
“Cytosolic viral sensor RIG-I is a 5'-triphosphate-dependent translocase on double-stranded
RNA.” Science. 323(5917):1070-4.

Gack MU, Kirchhofer A, Shin YC, Inn KS, Liang C, Cui S, Myong S, Ha T, Hopfner KP, Jung
JU (2008). “Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated
antiviral signal transduction.” Proc Natl Acad Sci U S A. 105(43):16743-8

Cui S, Eisenächer K, Kirchhofer A, Brzózka K, Lammens A, Lammens K, Fujita T,
Conzelmann KK, Krug A, Hopfner KP (2008). “The C-terminal regulatory domain is the RNA
5'-triphosphate sensor of RIG-I”. Mol Cell. (2):169-79

Parts of this thesis have been presented at an international conference:
Poster and talk “Structural and Functional Analysis of RIG-I Like Helicases” in the plenary
session at the Keystone Symposium Pattern Recognition Molecules and Immune Sensors of
th ndPathogens, March 29 – April 2 2009 in Banff, Alberta, Canada. TABLE OF CONTENTS
Part I - Structural and Functional Analysis of RIG-I Like Helicases
1 INTRODUCTION .................................................................................................. 1
1.1 Pattern Recognition Receptors in Innate Immunity .............................................................................. 1
1.2 RIG-I and MDA5: Virus Sensing and RNA Structures Recognized..................................................... 4
1.3 LGP2: A Regulator of RIG-I and MDA5 Signaling ............................................................................... 5
1.4 Downstream of RIG-I and MDA5: Transduction and Regulation of Antiviral Signaling .................. 6
1.5 Inhibition of RLR Signaling by Viral Proteins ....................................................................................... 8
1.6 Objectives................................................................................................................................................. 10
2 MATERIALS AND METHODS ............................................................................11
2.1 Materials ...................................................................................................................................................11
2.1.1 Chemicals.....................11
2.1.2 Bacterial Strains .................................................................................................................................11
2.1.3 Plasmids .............................................................................................................................................11
2.1.4 Media and Supplements .................................................................................................................... 12
2.1.5 Oligonucleotides ............................................................................................................................... 13
2.2 Methods.................................................................................................................................................... 14
2.2.1 Bioinformatic Methods........... 14
2.2.2 Molecular Biology Methods.............................................................................................................. 14
2.2.3 Cell Culture Methods ........................................................................................................................ 16
2.2.4 Protein Biochemistry Methods.......................................................................................................... 17
2.2.5 Functional Protein Assays ................................................................................................................. 18
2.2.6 Peptide SPOT Protein-Peptide Interaction Assay.............................................................................. 20
2.2.7 Structure Determination by Small Angle X-Ray Scattering.............................................................. 21
3 RESULTS 25
3.1 Structure Guided Mutagenesis of the RIG-I Regulatory Domain and RNA Binding Studies.......... 25
3.2 Functional Dissection of the Individual RIG-I Domains ..................................................................... 27
3.3 Functional Analysis of MDA5 and Its Interaction with Paramyxovirus V-Protein............................ 33
3.4 Structural Studies on the MDA5 Regulatory Domain with Small Angle X-Ray Scattering and
Homology Modeling................................................................................................................................. 38


4 DISCUSSION ..................................................................................................... 42
4.1 The Positively Charged Patch within RIG-I RD is the Recognition Site for 5’-Triphosphate RNA 42
4.2 RIG-I Integrates Two Pathogen-Associated Molecular Patterns........................................................ 43
4.3 CARDs Play a Dual Regulatory Role .................................................................................................... 43
4.4 Proposed Model for RIG-I Activation ................................................................................................... 44
4.5 Hypothetical Modes of Viral Recognition by MDA5............................................................................ 45
4.6 V-Protein Interferes with Helicase Activity of MDA5 and Thereby Inhibits Signaling .................... 46
4.7 MDA5 RD Structurally Resembles RIG-I RD...................................................................................... 46
5 SUMMARY ......................................................................................................... 48

Part II - Modulating Spectral Properties of the Green Fluorescent
Protein with Nanobodies
6 INTRODUCTION ................................................................................................ 51
6.1 Green Fluorescent Protein: From Initial Discovery to its Revolutionary Impact on Live Cell
Imaging..................................................................................................................................................... 51
6.2 Green Fluorescent Protein: Biophysical and Structural Properties ................................................... 52
6.3 Nanobodies as a Versatile Tool for Specific Protein Targeting ............................................................ 54
6.4 Structural Properties of Nanobodies ..................................................................................................... 56
6.5 Objectives................................................................................................................................................. 58
7 MATERIALS AND METHODS ........................................................................... 59
7.1 Materials .................................................................................................................................................. 59
7.1.1 Chemicals.................... 59
7.1.2 Bacterial Strains ................................................................................................................................ 59
7.1.3 Preparation of Minimal Medium for Selenomethionine Expression................................................. 59
7.2 Methods.................................................................................................................................................... 60
7.2.1