Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1 Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Universität Konstanz Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie Lehrstuhl für Biophysik Prof. Dr. Wolfram Welte vorgelegt von Diplom Biologe Mario Mörtl Referenten: Prof. Dr. Wolfram Welte (Konstanz) Prof. Dr. Peter Sonderegger (Zürich) Tag der mündlichen Prüfung, 8.12.2006 Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1 Index Zusammenfassung...............................................................................................................................1 Summary..............................................................................................................................................3 Abbreviations......................................................................................................................................5 A. Structure-function correlation of Glycine oxidase from Bacillus subtilis................................6 A.1. Abstract....................................................................................................................................7 A.2. Introduction..............................................................................................................................7 A.3. Experimental Procedures.........................................................................................................9 A.3.1. Growth conditions and preparation of cell extracts.........................................................9 A.3.2. Protein analyses, enzyme assay and gel-permeation chromatography............................9 A.3.3. Limited proteolysis experiments....................................................................................10 A.3.4. Preparation of the protein and crystallisation.................................................................10 A.3.5. Solution of the GO crystal structure...............................................................................11 A.3.6. Comparison of GR family members..............................................................................12 A.3.7. Accession numbers.........................................................................................................12 A.4. Results....................................................................................................................................12 A.4.1. Description of the structure............................................................................................12 A.4.2. Homology of GO with other amine oxidoreductases.....................................................17 A.4.3. FAD binding...................................................................................................................20 A.4.4. Mode of oligomerisation................................................................................................21 A.4.5. The active site................................................................................................................23 A.4.6. Effect of different carbon and nitrogen sources on B. subtilis growth and on GO expression..................................................................................................................................25 A.4.7. Effect of phosphate and thiamine pyrophosphate on GO activity.................................26 A.5. Discussion..............................................................................................................................27 B. Expression and refolding experiments of neuronal cell adhesion molecules.........................31 B.1. Introduction............................................................................................................................32 B.1.1. Axonal cell adhesion molecules.....................................................................................32 B.1.2. Chicken Axonin-1, NgCAM, and NrCAM guide the axons to the contralateral site.....33 B.1.3. Differences between chicken and rodent expression pattern.........................................34 B.1.4. L1 cell adhesion molecule..............................................................................................34 B.1.5. Chicken F11...................................................................................................................35 B.1.6. Chicken and human TAG-1...........................................................................................36 B.1.7. Immunoglobulin domains, properties and considerations..............................................36 B.1.8. Experimental goals.........................................................................................................37 B.2. Material and Methods............................................................................................................38 B.2.1. Chemicals and laboratory equipment.............................................................................38 B.2.2. Strains, cells , vectors, and primers................................................................................42 B.2.2.1. E. coli strains..........................................................................................................42 B.2.2.2. P. pastoris................................................................................................................43 B.2.2.3. Antibiotics...............................................................................................................44 B.2.3. Cloning...........................................................................................................................44 B.2.3.1. Colony PCR............................................................................................................44 P. pastoris........................................................................................................................44 III Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1 E. coli..............................................................................................................................44 B.2.3.2. Mutagenesis............................................................................................................44 B.2.3.3. Restriction enzymes................................................................................................45 B.2.3.4. Purification of DNA fragments..............................................................................45 B.2.3.5. DNA concentration.................................................................................................45 B.2.3.6. Ligation...................................................................................................................45 B.2.3.7. Chemically competent E. coli cells........................................................................45 Competent cells..............................................................................................................46 B.2.3.8. Transformation of E. coli cells...............................................................................46 B.2.3.9. Sequencing..............................................................................................................46 B.2.4. Protein analysis...............................................................................................................46 B.2.4.1. Protein concentration..............................................................................................46 Spectroscopy...................................................................................................................46 B.2.4.2. Electrophoresis.......................................................................................................47 SDS-PAGE.....................................................................................................................47 TCA precipitation...........................................................................................................47 Standard sample preparation...........................................................................................47 Refolding assay...............................................................................................................47 Isoelectric focusing.........................................................................................................47 Coomassie staining.........................................................................................................47 Silver staining.................................................................................................................48 B.2.5. Isolation of L1Ig1-4, NgCAMIg1-4, TAG-1Ig1-4, and E587-antigenIg1-4................................48 B.2.5.1. Expression medium................................................................................................48 B.2.5.2. Expression conditions.............................................................................................48 B.2.5.3. Isolation of inclusion bodies expressed in E. coli...................................................48 B.2.5.4. Affinity chromatography of denatured inclusion body proteins.............................49 B.2.6. Refolding........................................................................................................................49