Structural and Functional Studies Concerning Human N-Acetyl Mannosamine Kinase
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Structural and functional studies concerning human N-acetyl mannosamine kinase Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat) Eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin von Jacobo Martínez Font aus Barcelona, Spanien Berlin, August 2011 Die vorliegende Arbeit wurde in der Zeit von März 2008 bis August 2011 unter Anleitung von Prof. Dr. Saenger am Institut für Chemie und Biochemie/Kristallographie der Freien Universität Berlin im Fachbereich Biologie, Chemie, Pharmazie durchgeführt. 1. Gutachter: Prof. Dr. Wolfram Saenger 2. Gutachter: Prof. Dr. Udo Heinemann Disputation am: 10.11.2011 II Abbreviations Abbreviations Å Angstrom (10−10 m) ADP Adenosine diphosphate AMPPCP 5'‐adenylyl (beta,gamma‐methylene)diphosphonate AMPPNP Adenylyl Imidodiphosphate ATP Adenosine triphosphate bp base pair C‐terminal Carboxyl terminal CCD Charged coupled device CD circular dichroism CTP Cytidine triphosphate CV Column volume DANN Deoxyribonucleic acid DNAse Deoxyribonuclease EDTA Ethylenediaminetetraacetic acid GlcNAc N‐acetyl glucosamine GNE glucosamine (UDP‐ N‐acetyl)‐2‐epimerase/N‐acetylmannosamine kinase HIBM human inclusion body myopathy hMNK human N‐acetyl mannosamine kinase IPTG Isopropyl β‐D‐1‐thiogalactopyranoside ITC Isothermal titration calorimetry kDa Kilodalton LB‐Media Luria Bertani media M Molarity (mol/l) ManNAc N‐acetyl mannosamine ManNBut N‐butyl mannosamine ManNProp N‐propyl mannosamine mRNA messenger ribonucleic acid Mw molecular weight N‐terminal Amino terminal NeuNAc N‐acetyl neuraminic acid Ni‐NTA Ni2+‐nitrilotriacetic acid OD Optical density PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PDB Brookhaven Protein data bank PEG X Polyethylene glycol (mean molar mass X g / mol rmsd root mean square deviation RNA ribonucleic acid ROK Repressor, Open reading frame, kinase SDS Sodium dodecyl sulfate III Abbreviations TEMED N,N,N',N'‐Tetramethylethylenediamine TEV Tobacco etch virus Tris tris(hydroxymethyl)aminomethane UDP Uridine diphosphate UTP Uridine triphosphate UV Ultraviolet wt wild type % (v/v) volumen of solute with respect to the final volume of the solution % (w/v) x % (w/v) means x grams of solute per 100 ml solution IV Acknowledgments Acknowledgments First and foremost, I am heartily thankful to my advisor Prof. Wolfram Saenger for giving me the opportunity to work on such an interesting project, allowing me to work independently and develop my own ideas but guiding and supporting me continuously with patience. I kindly appreciate the fruitful discussions with him and his ability to make science, and more generally life, easy. I also want to thank Dr. Eva Tauberger, who carefully helped me to make the first steps on the way to this thesis. She has been always ready to help me in the lab and answering multitude of questions about molecular biology. Working together with her, preparing and supervising practical courses, has been a pleasure. I am very grateful to Dr. Sebastien Moniot for his incredible patience to answer all kind of crystallographic questions and for waking me up with good music every time I fell asleep during the night on our long beamtime measurements at the synchrotron. I say thank you to Sofia Banchenko, Dr.Sebastian Geibel, Alessandra Beltrami, Haydar Bulut, Traudy Wandersleben, Dr. Quingjun Ma, Dr. Albert Guskov, Dr. Ardeschir Vahedi, Dr. Azat Gabdulkhakov, Clemens Langner, Claudia Alings and Carsten Jakob who together with the above cited persons formed the AG Saenger and with whom I had great times that I will miss. Further, I am very thankful to Long Duc Nguyen, who realized most of the enzymatic and binding tests, for the nice and open cooperation. I acknowledge ditto Dr. Hua Fan and Prof. Dr. med. Werner Reutter, who started the project and developed the idea behind this thesis. I am grateful to all the members of the AG Wahl, AG Hauke and AG Scharf for their help during the hard laboratory move and reconstruction time. I am indebted to Dr. Chris Weise, who helpfully measured the MALDI-TOF spectra for different proteins and to Dr. Reinhold Zimmer for the synthesis of the hMNK Inhibitor. I am also thankful to Prof. Solioz, Dr. Helge Abitch and Dr. Steffano Mancini from the Swiss Copper Group for the good teamwork on the copper homeostasis project. Finally, I will always be thankful to my parents, family and friends for their unconditional support. V Index INDEX Abbreviations .................................................................................................................III Acknowledgments...........................................................................................................V 1. INTRODUCTION .....................................................................................................1 1.1. Contextualization of glycan and sialic acids .....................................................1 1.2. Sialic acids history and definition......................................................................2 1.3. Differential features of the sialic acid family .....................................................3 1.4. Sialic acids diversity .........................................................................................4 1.5. Sias general biological functions ......................................................................5 1.6. Sias recognition/antirecognition duality function...............................................6 1.7. The biosynthetic pathway of Sias in vertebrates ............................................10 1.8. The UDP-GlcNAc-2-Epimerase/ManNAc Kinase (GNE/MNK or GNE)..........12 1.9. Alternative roles of GNE.................................................................................14 1.10. GNE related diseases.................................................................................15 1.11. Previous available structural information on hGNE ....................................16 1.12. The MNK domain of GNE belongs to the ROK family ................................17 1.13. Starting point and aims of this thesis ..........................................................18 2. MATERIALS..........................................................................................................19 2.1. Instrumentation...............................................................................................19 2.2. Crystallization .................................................................................................20 2.3. Chemicals buffers and media.........................................................................21 2.4. Plasmids and bacterial strains........................................................................22 2.5. Primers ...........................................................................................................22 2.6. Enzymes, kits and ladders..............................................................................23 3. METHODS.............................................................................................................24 3.1. Cloning ...........................................................................................................24 3.1.1. Polymerase chain reaction (PCR) ...........................................................24 3.1.2. Agarose gel DNA extraction....................................................................25 3.1.3. DNA digestion .........................................................................................25 VI Index 3.1.4. DNA ligation ............................................................................................25 3.1.5. Preparation of chemically competent cells..............................................25 3.1.6. Transformation (Heat shock)...................................................................26 3.1.7. Colony PCR.............................................................................................26 3.1.8. Plasmid preparation ................................................................................26 3.1.9. DNA Sequencing.....................................................................................27 3.1.10. Clone and plasmid storage..................................................................27 3.1.11. In vitro DNA-Mutagenesis....................................................................27 3.2. Protein expression and purification ................................................................28 3.2.1. Expression test (small scale expression) ................................................28 3.2.2. Pre-culture...............................................................................................28 3.2.3. Protein overexpression............................................................................28 3.2.4. Cell disruption..........................................................................................29 3.2.5. Ultracentrifugation ...................................................................................29 3.2.6. Chromatographic purification ..................................................................29 3.2.6.1. Ni-NTA affinity chromatography...........................................................29 3.2.6.2. Size exclusion chromatography...........................................................30 3.2.7. hMNK digestion with TEV or Thrombin ...................................................30 3.3. General biochemical methods........................................................................30