Insights into the Mechanism of Action of the Protein Folding Nanomachine GroEL [Elektronische Ressource] / Manal Chatila. Betreuer: Ulrich Hartl

Manal Chatila

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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



Insights into the Mechanism of Action of the Protein
Folding Nanomachine GroEL







Manal Chatila
aus
Beirut, Libanon
2011 Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar
1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. Ulrich
Hartl betreut.















Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, 07/07/2011




(Unterschrift des Autors / der Autorin)









Dissertation eingereicht am 07/07/2011
1. Gutacher Prof. Ulrich Hartl
2. Gutachter PD Dr. Konstanze Winklhofer
Mündliche Prüfung am 04/08/2011 Acknowledgments
I am first and foremost greatly grateful for my supervisor Dr. Manajit Hayer-Hartl, whose
decision to accept me in the GroEL group has changed my life in ways I cannot start to describe,
both on the personal and professional levels. I am greatly in debt for Prof. Ulrich Hartl, who not
only offered unlimited resources to experiment and learn, but also allowed me to witness great
science in-the-making; I will miss those moments during lab meeting when I thought „Wow, that
is a great idea!“. Many thanks go to my thesis committee members for taking the time to evaluate
my work.
Great thanks go to the colleagues and friends who made my stay in the lab very enjoyable, and
more specifically the A1 office: Alex, Giulia, Florian, Paolo, Martin, Julien, Stephan, Roman,
and the recent additions who will further carry the GroEL flame Kristina, Amit and Javaid. I will
greatly miss our FACs (Friday Afternoon Cakes), and always cherish the great friendships I have
made.
Huge thanks go to the people without whom my research would not have been possible;
Bernhard, Guoxin, Sue, Jyoti and last but not least Kausik.
To the Hartl-fighters, I am thankful for all the late beergarten sessions, the bowling nights, the
get-togethers and all the laughs. Those always helped against research blues!
My research as well as my stay in Munich has always been very smooth and pleasant, thanks to
the great technical support from Emmanuel, Elizabeth, Silke, Andrea, Evelyn, Romy and
especially Nadine. Their willingness to help always brought in a breath of fresh air in the
department.
In the end, my deepest thanks and acknowledgments go to my parents for their endless support
and their trust in me. Your unlimited love gave me the strength to go on. Special thanks are due
to my brother Mohamed, for filling in my blanks. And last, great thanks go to Niclas for being
there, and for constantly encouraging and supporting. Without you my sun would not shine every
day.Contents i
Contents
1. Summary ................................................................................................................................ 1
2. Introduction ........................................................................................................................... 2
2.1 Protein folding ................................................................................................................ 2
2.1.1 Protein structure .............................................................................................. 2
2.1.2 The protein folding problem ............................................................................ 5
2.1.3 Methods to study protein folding .................................................................. 15
2.2 Protein folding in the cell .............................................................................................. 25
2.3 The cellular chaperone machinery ................................................................................ 26
2.3.1 Trigger factor ................................................................................................. 28
2.3.2 The DnaK/J system ....................................................................................... 29
2.3.3 GroEL/ES ...................................................................................................... 31
2.3.4 Additional E. coli chaperones ....................................................................... 41
2.4 Aim of the study............................................................................................................ 43
3. Materials and Methods ....................................................................................................... 44
3.1 Materials ....................................................................................................................... 44
3.1.1 Chemicals ...................................................................................................... 44
3.1.2 Enzymes ........................................................................................................ 46
3.1.3 Materials ........................................................................................................ 46
3.1.4 Instruments .................................................................................................... 47
3.1.5 Media ............................................................................................................. 48
3.1.6 Ampicillin stock solutions ............................................................................. 49
3.2 Bacterial strains and plasmids ....................................................................................... 49 Contents ii
3.2.1 E.coli strains .................................................................................................. 49
3.2.2 Plasmids ......................................................................................................... 49
3.3 Molecular cloning techniques ....................................................................................... 50
3.3.1 Preparation and transformation of E. coli competent cells............................ 50
3.3.2 Plasmid purification ...................................................................................... 51
3.3.3 PCR-mediated mutagenesis ........................................................................... 51
3.3.4 DNA analytical methods ............................................................................... 51
3.4 Protein purification ....................................................................................................... 51
3.4.1 GroEL expression and purification ............................................................... 51
3.4.2 GroES expression and purification ................................................................ 53
3.4.3 MBP and MBP mutants expression and purification .................................... 53
3.5 Protein analytical methods ............................................................................................ 54
3.5.1 Determination of protein concentration ........................................................ 54
3.5.2 SDS-PAGE .................................................................................................... 55
3.6 Biochemical and biophysical methods.......................................................................... 55
3.6.1 GroEL ATPase assay ..................................................................................... 55
3.6.2 In vitro MBP refolding assay ........................................................................ 56
3.6.3 Sample preparation for analysis of disulfide-bond formation in the cysteine
mutants .......................................................................................................... 57
3.6.4 Functional characterization of the cysteine mutants by maltose binding ...... 58
3.6.5 Unfolding and refolding curves of DM-MBP ............................................... 58
3.6.6 Typtophan fluorescence and Circular dichroism wavelength scans ............. 59
3.6.7 Unfolding kinetics by Stopped-flow measurements ..................................... 59
3.6.8 Static and Dynamic light scattering ............................................................... 59 Contents iii
3.7 Single-molecule FRET experiments ............................................................................. 60
3.7.1 Thiol-mediated labeling of the cysteine constructs ....................................... 60
3.7.2 Fluorescence correlation and cross-correlation spectroscopy ....................... 61
3.7.3 Single-pair FRET .......................................................................................... 63
3.8 Hydrogen/deuterium exchange experiments................................................................. 63
3.8.1 Pulse labeling ................................................................................................ 63
3.8.2 Mass spectrometric analysis .......................................................................... 64
4. Results .................................................................................................................................. 65