STRUCTURAL INVESTIGATIONS OF THE CANCER-ASSOCIATED LAMININ BINDING PROTEIN AND NOS L, A NOVEL COPPER BINDING PROTEIN by Lara Marie Taubner A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry MONTANA STATE UNIVERSITY Bozeman, Montana October 2005 COPYRIGHT by Lara Marie Taubner 2005 All Rights Reserved ii APPROVAL of a dissertation submitted by Lara Marie Taubner This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. Dr. Valérie Copié Approved for the Department of Chemistry and Biochemistry Dr. David Singel Approved for the College of Graduate Studies Dr. Joseph J. Fedock iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a doctorate’s degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non-exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Lara Marie Taubner October 2005 iv DEDICATION I would like to thank my mother, my father, and my sisters Kathleen and Sarah, for their unconditional love and support throughout these last years that has made this dissertation possible. v TABLE OF CONTENTS 1. INTRODUCTION ..........................................................................................................1 2. BACKGROUND ON THE LAMININ BINDING PROTEIN.......................................4 Carcinogenesis ................................................................................................................4 Extracellular Matrix-Cell Interactions and Tumorigenesis.............................................9 Laminin-1 and Peptide 11.............................................................................................10 The Laminin Binding Protein .......................................................................................13 LBP in Normal Tissues.................................................................................................16 The Laminin Binding Protein and Integrin...................................................................17 Characterization of Native Laminin Binding Protein ...................................................20 Ribosomal Function of LBP .........................................................................................23 The Laminin Binding Domain of LBP .........................................................................26 Hypothesis.....................................................................................................................28 2. MATERIALS AND METHODS FOR LBP ................................................................30 Cloning of Recombinant LBP in E.coli ........................................................................30 Expression and Purification of Bacterial Recombinant LBP........................................30 Limited Proteolysis and Identification of Domains......................................................31 Cloning and Expression of Proteolysis-derived Domains ............................................32 ELISA Assay and Laminin-1 Sepharose Affinity Chromatography ...........................................................................................................33 CD and NMR Spectroscopy .........................................................................................34 3. RESULTS FOR LBP ....................................................................................................36 Expression and Purification of Recombinant LBP .......................................................36 Biological Activity of Recombinant LBP.....................................................................40 Limited Proteolysis of Recombinant LBP ....................................................................40 Cloning and Expression of Proteolysis-derived Domains ............................................47 Biological Activity Screen of LBP Domains................................................................49 CD and NMR Spectroscopy of 200-295.......................................................................51 4. DISCUSSION ON LBP................................................................................................54 5. BACKGROUND ON NOS L .......................................................................................69 Denitrification...............................................................................................................69 Nitrous Oxide Reductase ..............................................................................................75 The nos Operon.............................................................................................................83 NosL..............................................................................................................................87 vi TABLE OF CONTENTS CONTINUED Metallochaperones ........................................................................................................91 Hypothesis.....................................................................................................................96 6. MATERIALS AND METHODS FOR NOS L ............................................................98 Expression and Purification of Recombinant Apo-NosL .............................................98 Expression and Purification of Recombinant Holo-NosL ............................................98 NMR Spectroscopy.......................................................................................................99 Structure Calculations.................................................................................................100 7. RESULTS FOR NOS L..............................................................................................106 Expression and Purification of Apo and Holo Nos L .................................................106 Sequential Assignment and Secondary Structure of Apo NosL .................................106 Apo NosL solution structure.......................................................................................116 Structure homologs to apo NosL ................................................................................129 Comparison of apo and holo 1H-15N HSQC spectra...................................................131 10. DISCUSSION ON NOS L........................................................................................139 11. CONCLUSIONS.......................................................................................................148 REFERENCES CITED....................................................................................................151 APPENDICES .................................................................................................................175 APPENDIX A. Complete Chemical Shift Assignments for Apo NosL ...................................................................................................................176 APPENDIX B. Amide proton and 15N data for apo vs holo NosL.........................................................................................................................189 APPENDIX C. Chemical shift file generated by NMRView........................................................................................................................195 APPENDIX D. Cross peak file generated by NMRView........................................................................................................................197 APPENDIX E. Conversion file for converting NMRView data into xml format......................................................................................199 APPENDIX F. ARIA project file, setup and run............................................................203 vii LIST OF TABLES Table Page 1. Fragments of LBP retrieved from limited proteolysis experiments......................46 2. NMR experiments and parameters......................................................................101 3. Restraints used in structure calculations and statistical analysis................................................................................................119 4. Residues exhibiting chemical shift differences between apo and holo NosL............................................................................................................136 viii LIST OF FIGURES Figure Page 1. Functional map of laminin...................................................................................12 2. Focal adhesion schematic ....................................................................................19 3. Amino acid sequence of the laminin binding protein ..........................................23 4. A. Sequence similarity between the N-terminal region of LBP to prokaryotic ribosomal proteins ......................................................25