Role of the MecA adaptor protein in regulation of the AAA_1hn+ chaperone ClpC of Bacillus subtilis [Elektronische Ressource] / presented by Tilman Schlothauer

Schlotha

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2004
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences







presented by
Diplom-Chemiker Tilman Schlothauer
from Hamburg






Oral examination:
1





Role of the MecA adaptor protein in
+regulation of the AAA chaperone ClpC
of Bacillus subtilis

















Referees: Prof. Dr. Bernd Bukau
Prof. Dr. Brunner

2 Table of Contents
TABLE OF CONTENTS
1A SUMMARY ........................................................................................................... 9
1B ZUSAMMENFASSUNG...................................................................................... 10
2 INTRODUCTION ................................................................................................... 11
2.1 Protein folding in vitro and in vivo.................................................................................. 11
2.2 The chaperone network 12
2.2.1 De novo folding........................................................................................................... 12
2.2.2 Repair of misfolded proteins....................................................................................... 13
2.2.3 Refolding of aggregated proteins ................................................................................ 14
2.3 The AAA+ superfamily.................................................................................................... 14
2.3.1 AAA+/HSP100 proteins involved in protein degradation ......................................... 16
2.3.2 Extra domains of AAA+/HSP100 proteins ................................................................. 17
2.4 AAA+ adaptor proteins ................................................................................................... 19
2.4.1 SspB and RssB ............................................................................................................ 19
2.4.2 ClpS............................................................................................................................. 20
2.5 AAA+ chaperones of Bacillus subtilis............................................................................. 21
2.6 Role of ClpC and the adaptor MecA in regulated and general proteolysis in B.
subtilis ...................................................................................................................................... 22
2.6.1 Regulated proteolysis in B. subtilis 22
2.6.1.1 Competence development..................................................................................... 22
2.6.1.2 The anti-sigma factor SpoIIAB is degraded by ClpCP ........................................ 23
2.6.1.3 ClpC and MecA degrade the transcriptional regulator Spx (YjbD) .................... 24
2.6.1.4 Degradation of MurAA by ClpCP........................................................................ 24
2.6.1.5 Degradation of CtsR............................................................................................. 24
2.6.2 General proteolysis in B. subtilis................................................................................. 25
2.7 Aim of this work ............................................................................................................... 26
3 Table of Contents
3 MATERIAL AND METHODS ................................................................................ 27
3.1 Materials ........................................................................................................................... 27
3.1.1 Equipment ................................................................................................................... 27
3.1.2 Chemicals.... 28
3.1.2 Standards and Kits....................................................................................................... 29
3.1.2.1 Protein standard................................................................................................... 29
3.1.2.2 Kits ....................................................................................................................... 29
3.1.3 Expendable items ........................................................................................................ 29
3.1.4 Proteins and Enzymes ................................................................................................. 30
3.1.5 Media and antibiotics .................................................................................................. 30
3.1.6 Materials for chromatography..................................................................................... 31
3.2 Molecular cloning techniques.......................................................................................... 31
3.2.1 Bacterial strains and plasmids 31
3.2.2 Competent cells of E. coli ........................................................................................... 33
3.2.3 Isolation of plasmid DNA from E. coli....................................................................... 34
3.2.3.1 Purification Protocol............................................................................................ 34
3.2.4 Cleavage of DNA with restriction endonucleases....................................................... 35
3.2.5 Gel electrophoresis of DNA........................................................................................ 35
3.3 Biochemical techniques.................................................................................................... 36
3.3.1 Preparation of ClpC-intein fusion proteins ................................................................. 36
3.3.1.1 Growth of bacteria ............................................................................................... 36
3.3.1.1.1 Purification of ClpC ...................................................................................... 36
3.3.1.1.2 Second purification step of ClpC .................................................................. 38
3.3.2 Preparation of His-tag fusion proteins ........................................................................ 39
3.3.2.1 Growth of bacteria 39
3.3.3 Other Proteins.............................................................................................................. 39
3.4 Gel electrophoresis and protein detection...................................................................... 40
3.4.1 Gel electrophoresis of proteins.................................................................................... 40
3.4.2 Coomassie staining...................................................................................................... 41
3.4.3 Silver stain................................................................................................................... 42
3.4.4 Immunological methods.............................................................................................. 43
3.4.4.1 Production of antisera: ........................................................................................ 43
4 Table of Contents
3.4.4.2 Immunoblotting:................................................................................................... 43
3.5 In vitro Activity Assays .................................................................................................... 45
3.5.1 ATPase assay............................................................................................................... 45
3.5.2 Prevention of aggregation and refolding of heat denatured Luciferase ...................... 46
3.5.3 Luciferase disaggregation and refolding..................................................................... 47
3.5.4 MDH disaggregation and refolding............................................................................. 47
3.5.5 Interaction of chaperones with MDH aggregates........................................................ 49
3.5.6 MDH/α-Casein degradation........................................................................................ 49
3.5.7 Analytical Size Exclusion Chromatography ............................................................... 49
33.5.8 H labeling of α-casein................................................................................................ 50
3.5.9 Cross-linking Assays................................................................................................... 51
3.5.10 BIAcore analysis ....................................................................................................... 52
3.5.11 Circular dichroism spectroscopy............................................................................... 53
3.6 In vivo activity assays 54
3.6.1 Gel filtration of cell extracts........................................................................................ 54
3.5.9.1 TCA precipitation................................................................................................. 55
3.6.2 Thermal resistance....................................................................................................... 55
3.6.2.1 Spot-tests ...........................