Protein folding in archaea [Elektronische Ressource] : analysis of the co-existing group I and group II chaperonins in M. mazei / Angela Maria Hirtreiter

Angela Hirtreiter

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 17 Mo

Extrait

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


Protein Folding in Archaea:
Analysis of the co-existing Group I and Group II
chaperonins in M. mazei



Angela Maria Hirtreiter
aus Straubing


München, 2006




Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. F.-Ulrich Hartl betreut.

Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 15.10.2006

…………………………..
(Angela Hirtreiter)







Dissertation eingereicht am 15.10.2006
1. Gutachter Prof. F.Ulrich Hartl

2. Gutachter PD Dr. Konstanze F. Winklhofer

Mündliche Prüfung am 31.01.2007



I
I. Summary .............................................................................................................................I-1
II. Introduction.........................................................................................................................II-1
II.1 Proteins......................................................................................................................II-1
II.1.1 Protein structure...............................................................................................II-1
II.1.2 Protein folding: from primary to quaternary structure....................................II-1
II.1.3 The protein folding mechanism.......................................................................II-2
II.2 Protein folding in vivo................................................................................................II-3
II.2.1 Protein folding in the cellular environment.....................................................II-3
II.2.2 Protein folding upon de novo synthesis .........................................................II-5
II.3 Molecular chaperones ..............................................................................................II-5
II.3.1 The chaperone system....................................................................................II-6
II.3.2 Ribosome associated chaperones .................................................................II-7
II.3.3 The Hsp70s ......................................................................................................II-8
II.3.4 Prefoldin / GimC...............................................................................................II-8
II.3.5 The chaperonins ..............................................................................................II-9
II.4 The Group I chaperonin system ........................................................................... II-11
II.4.1 Structure and function of the Group I chaperonin system GroEL/GroES. II-11
II.4.2 Mechanism of the GroEL/GroES system .................................................... II-12
II.4.3 Substrates of the GroEL/GroES system ..................................................... II-13
II.5 The Group II chaperonin system .......................................................................... II-14
II.5.1 Structure and function of Group II chaperonins.......................................... II-14
II.5.2 Mechanism of Group II chaperonins ........................................................... II-16
II.5.3 Substrates of Group II chaperonins............................................................. II-17
II.6 Archaea .................................................................................................................. II-18
II.6.1 The Methanogens......................................................................................... II-20
II.6.2 The genus Methanosarcina.......................................................................... II-21
II.6.3 Methanosarcina mazei Gö1 ......................................................................... II-23
II.7 Aim of the project ................................................................................................... II-24
III. Materials and Methods .................................................................................................. III-25
III.1 Materials ................................................................................................................ III-25
III.1.1 Chemicals..................................................................................................... III-25
III.1.2 Buffers and Media........................................................................................ III-27 _______________________________________________________________________II
III.2 Anaerobic cultivation............................................................................................. III-30
III.2.1 Set up of an anaerobic cultivation system ................................................. III-30
III.2.2 Anaerobic cultivation.................................................................................... III-31
III.2.3 Sterilisation................................................................................................... III-33
III.2.4 Light microscopy .......................................................................................... III-33
III.3 Molecular biochemical methods .......................................................................... III-34
III.3.1 Preparation and transformation of E. coli................................................... III-34
III.3.2 DNA analytical methods .............................................................................. III-34
III.3.3 DNA restriction/ligation methods ................................................................ III-35
III.3.4 Plasmid purification...................................................................................... III-35
III.4 Protein biochemical methods............................................................................... III-36
III.4.1 Protein expression and purification ............................................................ III-36
III.4.2 Protein analytical methods .......................................................................... III-38
III.4.3 Polyclonal antibodies................................................................................... III-42
III.4.4 Affinity purification of antibodies ................................................................. III-42
III.4.5 Western blotting ........................................................................................... III-43
III.4.6 Preparation of antibody beads.................................................................... III-43
III.4.7 Analytical gel filtration.................................................................................. III-43
III.5 Functional analysis methods................................................................................ III-44
III.5.1 Co-immunoprecipitations............................................................................. III-44
III.5.2 Substrate release experiments ................................................................... III-45
III.5.3 PK digestion ................................................................................................. III-45
III.6 Biophysical methods............................................................................................. III-45
III.6.1 Mass spectrometry of protein spots from 2D PAGE ................................. III-45
III.6.2 Coupled liquid chromatography – mass spectrometry system (LC-MS/MS)....
...................................................................................................................... III-46
III.7 Bioinformatic methods .......................................................................................... III-49
III.7.1 Sequence data analysis .............................................................................. III-49
III.7.2 Structure data analysis................................................................................ III-50
IV. Results ............................................................................................................................IV-51


_______________________________________________________________________III
IV.1 Production of recombinant proteins and polyclonal antibodies .........................IV-51
IV.2 Both chaperonins and their cofactors are expressed at similar levels and as
oligomeric proteins ............................................................................................. IV-53
IV.2.1 Group II chaperonin: The MmThs consists of three different subunits ....IV-53
IV.2.2 Group II chaperonin: the supposed co-chaperon prefoldin ......................IV-56
IV.2.3 Group I chaperonin MmGroEL and the Co-chaperone MmGroES ..........IV-59
IV.2.4 Both chaperonins are moderately induced under heat shock conditions IV-61
IV.3 Substrate identification .........................................................................................IV-62
IV.3.1 Co-precipitation of chaperonin-substrate complexes................................IV-62
IV.3.2 The MmGroEL/GroES complex is stable during the exper