Proteome-wide analyis of chaperonin-dependent protein folding in Escherichia coli [Elektronische Ressource] / Tobias Maier

Tobias Maier

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zu r Er langung de s D oktorgrades
der Fakultät für C hemie u nd P harmazie
der Lu dwig-Maximilians-Universität M ünchen
Proteome-Wide A nalysis of C haperonin-
Dependent P rotein F olding in Escherichia c oli
Tobias M aier
aus
Heilbronn
2006
Erklärung

Diese Dissertation wurde im Sinne von § 13 Absatz 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Professor Dr. F. Ulrich Hartl
betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig und ohne unerlaubte Hilfe erarbeitet.




München, 26. Oktober 2005





Dissertation eingereicht am 28. Oktober 2005
1. Gutachter: Professor Dr. F. Ulrich Hartl
2. Gutachter: PD Dr. Konstanze Winklhofer
Mündliche Prüfung am 01. Februar 2006
Contents I
1. Summary................................................................................ 1
2. Introduction................................. 2
2.1. From DNA to protein ............................................................................................ 2
2.1.1. Synthesis of proteins.................................................................................................................. 2
2.1.2. The protein folding problem.................................................................................................... 3
2.1.3. Protein folding mechanisms..................................................................................................... 5
2.1.4. Protein folding in vitro and in vivo .......................................................................................... 7
2.1.5. Diseases related to protein folding.......................................................................................... 8
2.2. Molecular chaperones............................................................................................ 9
2.2.1. Ribosome-associated chaperones .......................................................................................... 10
2.2.2. Hsp70 chaperones .................................................................................................................... 11
2.2.3. The reaction cycle of DnaK, DnaJ and GrpE ....................................................................... 13
2.3. Hsp60 and Hsp10: The chaperonins.................................................................. 13
2.3.1. E. coli chaperonins GroEL and GroES................................................................................... 14
2.3.2. The structure of GroEL and GroES ....................................................................................... 15
2.3.3. The mechanism of GroEL and GroES mediated protein folding..................................... 18
2.3.4. The substrates of GroEL and GroES ..................................................................................... 21
2.4. Chaperone networks in E. coli ............................................................................ 22
2.5. Introduction to proteomics ................................................................................. 24
2.5.1. Principles of mass spectrometry............................................................................................ 24
2.5.2. Technical possibilities and applications............................................................................... 24
3. Materials and Methods ...................................................... 26
3.1. Growth media and buffers.................................................................................. 26
3.1.1. Growth media........................................................................................................................... 26
3.1.2. Buffers and stock solutions.....................................................................................................26
3.2. Bacterial strains and plasmids............................................................................ 27
3.2.1. E. coli strains.............................................................................................................................. 27
3.2.2. Plasmids..................................................................................................................................... 27
3.3. DNA analytical methods..................................................................................... 28
3.3.1. PCR amplification .................................................................................................................... 28
3.3.2. DNA restriction, ligation and plasmid isolation................................................................. 29
3.4. Competent cells and transformation ................................................................. 30
3.5. Protein purification .............................................................................................. 31
3.6. Protein analytical methods ................................................................................. 32
3.6.1. Determination of protein concentration............................................................................... 32
3.6.2. SDS - PAGE ............................................................................................................................... 32
3.6.3. Silver staining ........................................................................................................................... 33
3.6.4. Western Blotting....................................................................................................................... 33
3.6.5. Generation of antibodies......................................................................................................... 34
3.6.6. Size exclusion chromatography............................................................................................. 34 Contents II
3.7. Protein refolding................................................................................................... 35
3.7.1. DAPA refolding........................................................................................................................ 35
3.7.2. DCEA refolding........................................................................................................................ 35
3.7.3. ENO refolding........................................................................................................................... 36
3.7.4. GATD refolding........................................................................................................................ 36
3.7.5. METF refolding......................................................................................................................... 36
3.7.6. METK refolding........................................................................................................................ 37
3.7.7. SYT refolding ............................................................................................................................ 37
3.8. In vivo co-expressions .......................................................................................... 37
3.8.1. Co-expressions of chaperones and substrates in E. coli ..................................................... 37
3.8.2. Co-expressions of chaperones anates in S. cerevisiae ......................................... 38
3.9. GroEL/GroES depletion ..................................................................................... 39
3.10. GroEL/GroES-substrate complexes........................................................... 39
3.10.1. Cell growth................................................................................................................................ 39
3.10.2. Cell lysis and purification of complexes .............................................................................. 40
3.10.3. Alternative purification method............................................................................................ 40
3.10.4. Proteinase K digestion of GroEL/GroES/substrate complexes....................................... 41
3.11. Mass spectrometric methods....................................................................... 41
3.11.1. Sample preparation for protein identification by mass spectrometry ............................ 41
3.11.2. Coupled liquid chromatography – mass spectrometry (LC-MS/MS) ............................ 42
3.11.3. Analysis of mass spectrometric data..................................................................................... 43
3.12. Bioinformatic methods................................................................................. 44
3.12.1. Structural comparison of GroE substrates ........................................................................... 44
3.12.2. Protein sequence analyses ...................................................................................................... 45
4. Results.................................................................................. 46
4.1. Identification of GroEL substrates ..................................................................... 46
4.1.1. Experimental approach ...........................................................................................................46
4.1.2. Stability of GroEL/GroES complexes................................................................................... 47