TECHNISCHE UNIVERSITÄT MÜNCHEN Department Chemie Lehrstuhl für Biotechnologie Analysis of small heat shock proteins and their interaction with substrate proteins Marina Angelika Kreuzeder 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. Vorsitzender: Prof. Dr. Bernd Reif Prüfer der Dissertation 1. Prof. Dr. Johannes Buchner 2. Prof. Dr. Michael Sattler Die Dissertation wurde am 18.09.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 02.11.2017 angenommen. Für meine Eltern Contents Contents 1. INTRODUCTION.............................................................................................. 1 1.1 Protein folding .................................................................................................................................. 1 1.2 Cell stress and heat shock proteins ........................................................................................... 3 1.3 Molecular chaperones .................................................................................................................... 5 1.4 ATP- dependent chaperones ........................................................................................................ 6 1.5 Small heat shock proteins ............................................................................................................. 9 1.5.1 Structure of Hsps ...................................................................................................................................... 9 1.5.2 Function of sHsps................................................................................................................................... 13 1.6 sHsps in different organisms ..................................................................................................... 15 1.6.1 sHsps in S. cerevisiae ............................................................................................................................. 16 1.6.2 human sHsps............................................................................................................................................ 18 1.6.3 sHsps in C. elegans ................................................................................................................................. 21 1.6.3.1 The Hsp16 core family ................................................................................................................ 23 1.7 Objectives .......................................................................................................................................... 26 2. MATERIAL ...................................................................................................... 27 2.1. Chemicals and media components .......................................................................................... 27 2.2 Consumables .................................................................................................................................... 29 2.3 Oligonucleotides ............................................................................................................................. 30 2.4 Substrate proteins ......................................................................................................................... 30 2.5 Antibodies ......................................................................................................................................... 31 2.6 Enzymes, standards and kits ...................................................................................................... 31 2.7 Strains ................................................................................................................................................ 32 2.8 Chromatography materials and columns .............................................................................. 33 2.9 Devices ............................................................................................................................................... 33 2.10 Software .......................................................................................................................................... 35 2.11 Databases ....................................................................................................................................... 36 I 2.12 Web-based tools ........................................................................................................................... 36 3. METHODS ...................................................................................................... 37 3.1 Molecular biology ........................................................................................................................... 37 3.1.1 Polymerase chain reaction ................................................................................................................ 37 3.1.2 Agarose gel electrophoresis ............................................................................................................... 38 3.1.3 Isolation of DNA fragments from Agarose gels .......................................................................... 39 3.1.4 Restriction digest and ligation .......................................................................................................... 39 3.1.5 Transformation of E.coli ...................................................................................................................... 40 3.1.6 Preparation of plasmid DNA from E. coli ...................................................................................... 40 3.1.7 DNA-sequence analysis ........................................................................................................................ 40 3.2 Protein analytics ............................................................................................................................. 41 3.2.1 Determination of protein concentration ....................................................................................... 41 3.2.1.1 Bradford-Assay ............................................................................................................................... 41 3.2.1.2 BCA-Assay ......................................................................................................................................... 41 3.2.2 SDS-Polyacrylamid-Gelelectrophoresis (SDS-PAGE) ............................................................... 42 3.2.3 Staining of SDS-PAGE gels according to Fairbanks ................................................................... 43 3.2.4 Western-blotting ..................................................................................................................................... 43 3.3 Preparative methods .................................................................................................................... 45 3.3.1 Cell disruption .......................................................................................................................................... 45 3.3.2 Concentration of proteins ................................................................................................................... 45 3.3.3 Dialysis of proteins ................................................................................................................................ 45 3.4 Chromatography ............................................................................................................................. 46 3.4.1 Ion exchange chromatography ......................................................................................................... 46 3.4.2 Size exclusion chromatography ........................................................................................................ 46 3.5 Expression and Purification of Proteins ................................................................................ 47 3.5.1 Expression and purification of CeHsp16 proteins .................................................................... 47 3.5.2 Expression and purification of human αB- crystallin .............................................................. 49 3.5.3 Expression and purification of ScHsp26wt and truncation mutants ................................ 50 3.6 Spectroscopical methods ............................................................................................................. 53 3.6.1 UV/VIS-Spectroscopy ............................................................................................................................ 53 3.6.2 Chaperone activity assays ................................................................................................................... 54 3.6.3 CD-Spectroscopy ..................................................................................................................................... 55 3.6.4 Analytical Size Exclusion Chromatography ................................................................................. 56 3.6.5 Analytical Ultracentrifugation ........................................................................................................... 57 3.6.6 Electron Microscopy ............................................................................................................................. 58 3.7 Mass spectrometry ........................................................................................................................