Institut für Organische Chemie und Biochemie Lehrstuhl für Biotechnologie Characterization of the DnaK-DnaJ-GrpE system under oxidative heat stress Katrin Linke Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzende: Univ.-Prof. Dr. S. Weinkauf Prüfer der Dissertation: 1. Univ.-Prof. Dr. J. Buchner 2. Asst.-Prof. U. Jakob, Ph.D., University of Michigan, USA Die Dissertation wurde am 17.01.2005 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 17.02.2005 angenommen. i Contents 1 SUMMERY...........................................................................................................1 2 INTRODUCTION..................................................................................................3 2.1 About the Ups and Downs of proteins ........................................................................3 2.1.1 Protein folding in vivo ............................................................................................3 2.1.2 Chaperones – Helpers in hard times.......................................................................4 2.1.3 The many classes of molecular chaperones............................................................5 2.2 Heat shock response and its regulation ......................................................................7 2.3 The DnaK/DnaJ/GrpE-system ....................................................................................8 2.3.1 The molecular chaperone DnaK.............................................................................9 2.3.2 The molecular chaperone DnaJ ............................................................................10 2.3.3 Mechanism of the DnaK/DnaJ/GrpE-system .......................................................13 2.4 Aerobic life and oxidative stress................................................................................14 2.4.1 Redox regulation of protein activity.....................................................................17 2.5 Objective......................................................................................................................21 3 MATERIAL AND METHODS .............................................................................22 3.1 Material .......................................................................................................................22 3.1.1 Strains...................................................................................................................22 3.1.2 Plasmids................................................................................................................22 3.1.3 Primer ...................................................................................................................23 3.1.3.1 Mutagenesis Primer ..........................................................................................23 3.1.3.2 Sequencing Primer............................................................................................23 3.1.4 Proteins.................................................................................................................23 3.1.5 Antibodies, Markers, Dyes, Antibiotics and Inhibitors........................................24 3.1.6 Chemicals .............................................................................................................24 3.1.7 Buffers and Solutions ...........................................................................................26 3.1.8 Kits and Chromatography Material ......................................................................27 3.1.9 Other Material......................................................................................................27 3.1.10 Technical Equipment............................................................................................28 3.1.11 Software................................................................................................................29 3.2 Molecular-biological methods ...................................................................................29 3.2.1 Cultivation and conservation of E. coli strains.....................................................29 3.2.2 QuikChange site-directed mutagenesis ................................................................29 3.2.2.1 Construction of DnaJ zinc center mutants........................................................30 3.2.2.2 Construction of the DnaKCys15Ala-mutant..........................................................31 3.2.3 Preparation and transformation of heat competent cells ......................................31 3.3 Preparative methods ..................................................................................................31 3.3.1 Ammonium sulfate precipitation..........................................................................32 3.3.2 Chromatography ...................................................................................................32 ii 3.3.3 Concentration and dialysis ...................................................................................33 3.3.4 Purification of ATP-depleted DnaK.....................................................................34 3.3.5 Purification of DnaJ..............................................................................................35 3.3.6 Purification of GrpE.............................................................................................37 3.4 Protein-biochemical methods ....................................................................................38 3.4.1 Determination of protein concentration................................................................38 3.4.2 Oxidation and reduction of DnaJ..........................................................................39 3.4.3 Inactivation and reactivation of DnaK in vitro.....................................................40 3.4.3.1 Oxidation of DnaK ...........................................................................................40 3.4.3.2 Reduction of DnaK...........................................................................................40 3.4.4 Determination of free thiol groups in proteins .....................................................40 3.4.4.1 Ellman’s assay..................................................................................................40 3.4.4.2 PAR-PMPS assay.............................................................................................41 3.4.4.3 AMS trapping of DnaJ......................................................................................41 3.4.4.4 Biotinylation of DnaK ......................................................................................42 3.4.5 SDS PAGE and Protein Staining..........................................................................43 3.4.6 Immunoblotting (western blotting).......................................................................44 3.5 Spectroscopic methods ...............................................................................................45 3.5.1 Fluorescence Measurements of DnaK..................................................................45 3.5.2 Circular dichroism measurements ........................................................................45 3.6 Chaperone assays in vitro...........................................................................................46 3.6.1 Light scattering experiments ................................................................................46 3.6.1.1 Aggregation of chemically denatured luciferase..............................................47 3.6.1.2 Aggregation of chemically denatured citrate synthase.....................................47 3.6.2 Reactivation of chemically denatured luciferase..................................................47 3.6.3 Reactivation of chemically denatured citrate synthase.........................................48 3.6.4 Determination of DnaK’s ATPase activity...........................................................48 3.6.4.1 Steady state ATPase assay................................................................................49 3.6.4.2 Single turn over ATPase assay.........................................................................49 3.7 Phenotypic assays and other in vivo methods ..........................................................49 3.7.1 Growth under stress conditions ............................................................................49 3.7.2 Cell motility assay ................................................................................................50 3.7.3 Phage λ replication assay .....................................................................................50 3.7.4 Determination of cellular ATP level ....................................................................50 4 RESULTS ..........................................................................................................52 4.1 Characterization of DnaJ under oxidative stress conditions..................................52 4.1.1 DnaJ – Chaperone with two zinc centers .............................................................52 4.1.2 Zinc content of DnaJ in vitro................................................................................52 4.1.3 Oxidation of DnaJ in vitro....................................................................................53