The HSP47 - Procollagen Interaction: Mechanism of pH-Dependent Client Release and Development of Antifibrotic Inhibitors Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakültät der Universität zu Köln Vorgelegt von Sinan Öcal aus Istanbul Köln, September 2018 Gutachter: Prof. Dr. Ulrich Baumann Institut für Biochemie / Universität zu Köln Prof. Dr. Karsten Niefind Institut für Biochemie / Universität zu Köln Prüfungsvorsitzender: Prof. Dr. Eric von Elert Institut für Zoologie / Universität zu Köln Tag der mündlichen Prüfung: 13. 11. 2017 Die Arbeiten und Experimente zur vorliegenden Dissertation wurden zwischen Oktober 2012 und September 2017 unter Betreuung von Prof. Dr. Ulrich Baumann am Institut für Biochemie der Universität zu Köln, Otto-Fischer Str. 12-14, D-50674 Köln, Deutschland, durchgeführt. Teile dieser Arbeit wurden bereits verö ffentlicht: Oecal S, Socher E, Uthoff M, et al. The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair. The Journal of Biological Chemistry . 2016;291(24):12612-12626. Table of Contents Zusammenfassung ........................................................................................................................ i Abstract ...................................................................................................................................... iii 1. Introduction ............................................................................................................................. 1 1.1 Collagen ............................................................................................................................. 1 1.1.1 The Collagen Superfamily: An Overview ................................................................................ 1 1.1.2 Composition and Classification of Collagens ......................................................................... 2 Fibril Forming Collagens .............................................................................................................. 3 1.1.3 Structure and Stability of the Collagenous Domain ............................................................... 4 The Polyproline Helix Type II ....................................................................................................... 4 The Collagen Triple-helix ............................................................................................................. 5 Structural Aspects of Triple-helix Stability .................................................................................. 5 1.1.4 Collagen Biosynthesis ............................................................................................................. 9 Hydroxylation .............................................................................................................................. 9 Glycosylation ............................................................................................................................. 10 Cis-trans isomerization ............................................................................................................. 10 Folding of collagens .................................................................................................................. 10 Trafficking, processing and secretion ....................................................................................... 11 1.2 Heat Shock Protein 47 ....................................................................................................... 14 1.2.1 An Introduction to HSP47 .................................................................................................... 14 1.2.2 Serpins and Structure of HSP47 ........................................................................................... 15 1.2.3 HSP47 in Collagen Biosynthesis ........................................................................................... 17 1.2.4 Molecular details of the HSP47 - Collagen Interaction ........................................................ 18 1.3 Thesis Aims ...................................................................................................................... 20 Molecular details of the pH-dependent client-release ............................................................. 20 Identifying small organic molecule inhibitors of the HSP47 - collagen interaction .................. 21 2. Material and Methods ............................................................................................................ 22 2.1 Material ........................................................................................................................... 22 2.1.1 Chemicals ............................................................................................................................. 22 2.1.2 Proteins ................................................................................................................................ 22 2.1.3 Buffers and Media ................................................................................................................ 23 2.1.4 Synthetic Peptides................................................................................................................ 24 2.1.5 Synthetic DNA Oligonucleotides .......................................................................................... 24 2.1.6 E. coli Strains ........................................................................................................................ 24 2.1.7 Software ............................................................................................................................... 24 2.2 Nucleic Acid Methods ....................................................................................................... 24 2.2.1 Isolation & Purification of DNA ............................................................................................ 24 2.2.2 Spectrometric Determination of DNA concentration .......................................................... 25 2.2.3 Agarose Gel Electrophoresis ................................................................................................ 25 2.2.4 Polymerase Chain Reaction & Site-directed Mutagenesis ................................................... 26 2.2.5 Restriction Endonuclease Digestion of DNA & Ligation ....................................................... 27 2.2.6 DNA Sequencing ................................................................................................................... 28 2.3 Cell Culture Methods ........................................................................................................ 28 2.3.1 Transformation of DNA ........................................................................................................ 28 2.3.2 Protein Expression in E. coli ................................................................................................. 28 2.3.3 Cell lysis ................................................................................................................................ 29 2.4 Protein Biochemistry Methods .......................................................................................... 29 2.4.1 Chromatographic Methods .................................................................................................. 29 2.4.2 SDS-PAGE ............................................................................................................................. 31 2.4.3 Western Blotting & Immunodetection ................................................................................ 32 2.4.4 Biotinylation of Proteins ...................................................................................................... 33 2.4.5 Spectrometric Determination of Protein concentration ..................................................... 33 2.4.6 Colorimetric Protein Assays ................................................................................................. 33 2.4.7 Differential Scanning Fluorimetry ........................................................................................ 34 2.4.8 Circular Dichroism Spectroscopy ......................................................................................... 34 2.4.9 Isothermal Titration Calorimetry ......................................................................................... 35 2.4.10 Dynamic Light Scattering ................................................................................................... 35 2.4.11 Biolayer Interferometry ..................................................................................................... 36 2.4.12 Fluorescence Spectroscopy & Homogenous Time-Resolved FRET .................................... 36 2.4.13 Protein Crystallization ........................................................................................................ 39 3. Results ................................................................................................................................... 40 3.1 Protein Production and Characterization ........................................................................... 40 3.1.1 Proteins and Peptides used in this Work ............................................................................. 40 HSP47 Constructs ...................................................................................................................... 40 Collagen Model Peptides .........................................................................................................