Crystallization trials of refolded breast tumor kinase (BRK) and peroxisome proteins 1

ACKNOWLEGEMENTS This thesis would not have been accomplished without the invaluable help of Professor Kunchithapadam SWAMINATHAN. I have been privileged to be a graduate student in his lab and work with two interesting projects. I thank him for encouraging me with the development of these projects, the lectures taught on X-ray diffraction, correcting my thesis, along with many good suggestions and for his sense of humor, patience and professionalism. I would also like to acknowledge his help on the chance to work in Prof. Suresh Subramani’s lab at University of California, San Diego, USA where I have learned protein expression in yeast and other related techniques. I would like to acknowledge my lab members who helped me complete this thesis, in particular: Pankaj for his invaluable helps and discusses about how to use the FPLC machine and how to interpret results; Anupama, Kuntal and Vindhya for helping me understand protein purification and crystallization; Shiva for teaching me cloning; and other lab members, Umar, Fengxia and Kanmani for many great discussions on research. I also thank the lab member, Abilash, Lissa, Manjeet, Nilofer, Thangavelu, Veeru and Tzer Fong, of Dr. Sivaraman’s lab for their support in protein expression, extraction and purification to complete my research. Special thank to Dr. Sivaraman for allowing me to use the dynamic light scattering machine and other equipments. I would like to acknowledge his recommendation letters and encouragement. I am very grateful to Prof. Subramani and all his lab members, who helped me complete the peroxisome projects. Special thanks to Dr. Changle Ma, who made the i    peroxisome constructs and helped me all the times in USA. Most importantly, I would like to thank to Prof. Suresh Subramani for giving me the wonderful opportunity to work in his lab and learn many interesting techniques. I value his support, encouragement, and the time he provided to know the US culture. I would specially like to thank Karthik (SBL2, DBS) and Rishi (SBL1, DBS) who taught me how to use the CD spectrum and fluorescence machine. I also enjoyed talking to them and learned so much about these experiments. I also thank them for all their suggestions on my Pre-Thesis report and seminar. I would like to thank NUS for the research scholarship and supporting me to finish my Masters degree. I also thank to Department of Biological Sciences for giving me all best conditions and preparations, from lectures, conferences to my PreThesis presentation. I would specially like to thank Ms. Reena for giving me all the necessary information, always. I also thank all professors for teaching several wonderful modules and interesting techniques. For my personal notes, I would like to thank my girl friends and family that have supported me throughout my course of study. I thank my girlfriend for her invaluable encouragement and advice. She has kindly takes care of my family and I share my life with her confident smile. Most of all, as always, I thank my family, Mum, Dad and Thang (my younger brother) for supporting me in every way. They are the proof that some of us are just born lucky. Finally, I would like to thank the two Pre-thesis examiners, Prof. Maxey Chung (from Biochemistry, NUS) and Prof. Kanagasabapathy (from National Cancer Center, Singapore) for their time to read through my presentation and give me much ii    critical suggestions. Following their recommendations, I mainly present the refolded Brk part for my Mater thesis along with limited highlights of the peroxisome proteins in my thesis. However, I expect that both these projects promise interesting publications. iii    TABLE OF CONTENTS Page Acknowledgements i Table of contents iv Abstract ix List of abbreviations xi List of figures xiv CHAPTER 1 MACROMOLECULAR X-RAY CRYSTALLOGRAPHY 1.1 PROTEIN STRUCTURE DETERMINATION 1 1.2 PROTEIN CRYSTALLOGRAPHY 2 1.2.1 X-ray crystallography for proteins 2 BASIC CONCEPTS IN CRYSTALLOGRAPHY 3 1.3.1 3 1.3 Unit cell and lattices 1.3.2 Symmetry, point group and space group 4 1.3.3 Crystals and X-rays 5 1.3.4 X-ray diffraction 5 1.3.5 Bragg’s law 6 1.3.6 Reciprocal lattice and Ewald sphere 7 1.3.6.1 Ewald sphere 7 Fourirer transform, structure factor and phase problem 7 1.3.7 1.4 DATA COLLECTION 8 1.5 STRUCTURE DETERMINATION 9 iv    1.5.1 Phasing techniques 1.5.2 1.5.3 9 1.5.1.1 Direct method 9 1.5.1.2 Molecular replacement 9 1.5.1.3 Multiwavelengh isomorphous replacement 10 1.5.1.4 Anomalous dispersion 10 Model building and refinement 11 1.5.2.1 R Factor 11 Validation and presentation 11 1.5.3.1 Ramachandran Plot 12 1.5.3.2 Folding profile methods 12 CHAPTER 2 BIOLOGY BACKGROUND 2.1 2.2 BREAST TUMOR KINASE (BRK) 13 2.1.1 Protein tyrosine kinase in signal transduction 13 2.1.2 Brk family non-receptor tyrosine kinases 15 2.1.3 Brk 16 2.1.4 Role of Brk in lymphoma 20 THE IMPORTANT ROLES OF PEX8 AND PEX20 IN PICHIA PASTORIS 23 2.2.1 Peroxisome biogenesis and degradation 23 2.2.1.1 Peroxisomal constituents and its functions 23 2.2.1.2 Basic views of Lipid and Protein Import into Peroxisomes 24 2.2.1.3 Components of the peroxisomal matrix and membrane protein 2.2.2 import machinery 25 Peroxisomes and human diseases 29 v    2.2.3 2.3 2.2.2.1 Peroxisome biogenesis disorders 29 2.2.2.2 Peroxisomal single protein defects 30 2.2.2.3 Diagnosis and therapy 30 Pichia pastoris Pex8 and Pex20: role in peroxisomal matrix protein import machinery 31 2.2.3.1 Pichia pastoris Pex8 (Pex8) 31 2.2.3.2 Pichia pastoris Pex20p (Pex20) 32 OBJECTIVE 32 CHAPTER 3 MATERIALS AND METHODS 3.1 3.2 EXPRESSION AND PURIFICATION OF RECOMBINANT WT-BRK 34 3.1.1 Construction of expression for WT-Brk 34 3.1.2 Bacterial expression 35 3.1.3 Solubilization, purification and refolding from inclusion body 36 3.1.3.1 Solubilization 36 3.1.3.2 Purification 36 3.1.3.3 Rapid dilution 36 3.1.3.4 Size-exclusion chromatography 37 3.1.3.5 Western blot analysis for denatured and refolded WT-Brk 37 3.1.3.6 Dynamic light scattering 38 3.1.3.7 Circular dichroism spectroscopy 38 3.1.3.8 Intrinsic tryptophan fluorescence measurement 39 3.1.3.9 Crystallization 39 EXPRESSION AND PURIFICATION OF RECOMBINANT GST-Pex8 AND GST-Pex20 39 3.2.1 39 Bacterial expression vi    3.2.2 Affinity purification of GST-Pex8 and GST-Pex20 40 3.2.3 Anion exchange chromatography 41 3.2.4 Gel filtration 41 3.2.5 Mass spectroscopy identity for GST-Pex8 and GST-Pex20 proteins 41 3.2.6 Protein concentration identification 42 CHAPTER 4 RESULTS AND DISCUSSION 4.1 4.2 WT-BRK REFOLDING, STRUCTURE ANALYSIS AND CRYSTALLIZATION 43 4.1.1 Bacterial expression 43 4.1.2 WT-Brk affinity purification 44 4.1.3 Purification of refolded WT-Brk by gel filtration 45 4.1.4 The homogeneity of refolded Brk 46 4.1.5 Characterization of refolded Brk by Western blot 48 4.1.6 Circular Dichroism (CD) of WT-Brk 49 4.1.7 Intrinsic tryptophan fluorescence spectroscopy for refolded Brk 52 4.1.8 Crystallization of refolded Brk 55 PEROXISOME PROTEIN EXPRESSION, PURIFICATION CRYSTALLIZATION 4.2.1 AND 56 Bacterial expression and affinity purification for GST-Pex8 (peroxin factor 8) and GST-Pex20 (peroxin factor 20) constructs 56 4.2.2 Gel filtration chromatography for peroxisome proteins 58 4.2.3 Optimized purification for GST-Pex 20 using anion exchange column 58 CHAPTER 5 CONCLUSION AND FUTURE STUDIES 62 vii    5.1 CONCLUSION 62 5.2 FUTURE STUDIES 63 REFERENCES 65 APPENDICES viii    ABSTRACT Protein expression and purification are essential steps and initial conditions for crystallization works and crystal structure studies. Here, we are presenting two different parts that is much relative to the protein expression and purification experiments. The first part, also a major project, is focusing on the preliminary crystallization trials of refolded Breast tumor kinase (Brk). Breast tumor kinase (Brk), a non-receptor tyrosine kinase that is overexpressed in a high percentage of breast carcinomas, has been found to be constitutively expressed in a large proportion of cutaneous T-cell lymphomas and other transformed T- and B-cell populations. Brk also has conferred in vivo oncogenicity on the BaF3 cells. Furthermore siRNA-mediated inhibition of endogenous Brk in malignant T-cells has diminished their growth and survival capacity. However, the role of Brk in cell transformation remains poorly defined. To determine the actual mechanism that is responsible for the Brk-mediated control over basic cell functions, we propose to elucidate the three-dimensional structure of Brk using X-ray crystallography and correlate with its function. The structure of Brk (and its mutants) will eventually help to identify potential therapeutic targets for lymphomas and, possibly, other malignancies. Furthermore, studies are underway to identify the interacting partners of Brk in lymphoma. The recombinant human Brk protein was expressed in bacteria in the insoluble form and refolded. The refolded protein was characterized by several biophysical methods, like, intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The second part is mainly concerning on expression and purification of peroxins (peroxisome factor 8 and peroxisome factor 20). Peroxins are proteins that are required for various aspects of peroxisome biogenesis including assembling ix    peroxisome membrane, importing most of the peroxisomal matrix proteins, peroxisome proliferation and peroxisome inheritance. Two of such peroxins, Pex8 and Pex20 that are involved in Peroxisome targeting signal 2 (PTS2) pathway, are known to play key roles in transporting important peroxisomal matrix proteins into the matrix of the peroxisome. These proteins also interact with the Pex7 receptor to translocate proteins that have the PTS2 sequence into the peroxisome. Even though most of the activities and functions of these proteins have been predicted, full understanding of these proteins will be possible after theirs structure are known. In the present study, we have successfully over-expressed and purified GST-Pex8 and GST-Pex20. x    LIST OF ABBREVIATIONS AAA-ATPases ATPases Associated with various cellular Activities ABC ATP-binding cassette ALK anaplastic lymphoma kinase ATP Adenosine diphosphate BRK Breast tumor kinase Bsk β-cell Src-homology tyrosine kinase CCD Charged coupled device CTCL Cutaneous T cell lymphoma DLS Dynamic light scattering EGF Epidermal growth factor EGFR Epidermal growth factor receptor ErbB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) Frk Fyn-related kinase Gtk Gut tyrosine kinase HEK Human Embryonic Kidney IRD Infantile Refsum disease xi    Iyk Intestinal tyrosine kinase MAD Multi wavelength anomalous dispersion mPTSs Membrane peroxisome targeting signal Myr Myristoylation NALD Neonatal adrenoleukodystrophy NMR Nuclear Magnetic Resonance NOE Nucleus of normal oral epithelium OSCC Oral squamous cell carcinomas PBDs Peroxisome biogenesis disorders PBMC Peripheral blood Mononuclear Cell PDB Protein Data Bank PEX Peroxisome PTK6 Protein tyrosine kinase 6 PTKs Protein tyrosine kinases PTS1 Peroxisome targeting signal 1 PTS2 Peroxisome targeting signal 2 RADAR The peroxisomal receptor accumulation and degradation in the absence of recycling Rak Also named GTK/Bsk/IYK xii    SAD Single wavelength anomalous dispersion SH2 Src homology 2 domain SH3 Src homology 3 domain Sik salt-inducible kinase Src Sarcoma Src42A Src oncogene at 42A Srms Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites UBC Ubiquitin-conjugating protein VLCFA Unbranched very long chain fatty acids XALD X-linked adrenoleukodystrophy YP Phosphotyrosine residues ZS Zellweger syndrome xiii    LIST OF FIGURES Figure 1.1: A single protein crystal 3 Figure 1.2: Unit-cells and 14 Bravais lattices 4 Figure 1.3: The two types of interference 6 Figure 1.4: Ewald Sphere and radius equation 7 Figure 1.5: X-ray diffractometer 9 Figure 2.1: Structure of Src and Brk tyrosine kinases 19 Figure 2.2: Electron micrograph of rat liver 24 Figure 2.3: Peroxisome morphology on different growth media (Pichia pasteris) 25 Figure 2.4: Targeting signals used by peroxisomal proteins 26 Figure 2.5: Membrane protein complexes of the peroxisomal protein import machinery 27 Figure 2.6: Models for the role of ubiquitylation in receptor recycling (or dislocation) from the peroxisome membrane to the cytosol, and in degradation by the RADAR pathway 29 Figure 4.1: Overexpression of Brk 43 Figure 4.2: WT-Brk was over-expressed in inclusion body after 3 hours induction 44 Figure 4.3: Purification of Brk 44 Figure 4.4: Size-exclusion chromatography profile of refolded WT-Brk 45 Figure 4.5: The homogenecity of refolded Brk 46 xiv    Figure 4.6: The dispersity characteristic of refolded Brk using Dynamic Light Scattering (DLS) 47 Figure 4.7: DLS profile for refolded Brk at 3 mg/mL concentration 48 Figure 4.8: Western blot analysis of Brk refolding 49 Figure 4.9: A far-UV CD spectrum for refolded WT-Brk 51 Figure 4.10: The CD-spectra of WT-Brk at different temperatures 51 Figure 4.11: Thermal denaturation scanning for refolded WT-Brk 52 Figure 4.12: The intrinsic tryptophan fluorescence changes of WT-Brk when thermally unfolded 54 Figure 4.13: Intrinsic tryptophan fluorescence under chemical stress with urea (2 and 4 M) 55 Figure 4.14: Overexpression of GST-Pex20 and GST-Pex8 in BL21 (DE3) E. coli cells 56 Figure 4.15: Optimized purification of GST-Pex8 by lysozyme addition via affinity chromatography using GSTrap FF 1 mL from GE Healthcare 57 Figure 4.16: Gel filtration profile for GST-Pex8 59 Figure 4.17: Anion exchange purification of GST-Pex20 61 Figure 4.18: Peptide mass fingerprinting of GST-Pex8 62 Figure 4.19: Peptide mass fingerprinting of GST-Pex20 62 xv    [...]... homogenecity of refolded Brk 46 xiv    Figure 4.6: The dispersity characteristic of refolded Brk using Dynamic Light Scattering (DLS) 47 Figure 4.7: DLS profile for refolded Brk at 3 mg/mL concentration 48 Figure 4.8: Western blot analysis of Brk refolding 49 Figure 4.9: A far-UV CD spectrum for refolded WT-Brk 51 Figure 4 .10 : The CD-spectra of WT-Brk at different temperatures 51 Figure 4 .11 : Thermal... interference 6 Figure 1. 4: Ewald Sphere and radius equation 7 Figure 1. 5: X-ray diffractometer 9 Figure 2 .1: Structure of Src and Brk tyrosine kinases 19 Figure 2.2: Electron micrograph of rat liver 24 Figure 2.3: Peroxisome morphology on different growth media (Pichia pasteris) 25 Figure 2.4: Targeting signals used by peroxisomal proteins 26 Figure 2.5: Membrane protein complexes of the peroxisomal protein... 4 .11 : Thermal denaturation scanning for refolded WT-Brk 52 Figure 4 .12 : The intrinsic tryptophan fluorescence changes of WT-Brk when thermally unfolded 54 Figure 4 .13 : Intrinsic tryptophan fluorescence under chemical stress with urea (2 and 4 M) 55 Figure 4 .14 : Overexpression of GST-Pex20 and GST-Pex8 in BL 21 (DE3) E coli cells 56 Figure 4 .15 : Optimized purification of GST-Pex8 by lysozyme addition via... Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites UBC Ubiquitin-conjugating protein VLCFA Unbranched very long chain fatty acids XALD X-linked adrenoleukodystrophy YP Phosphotyrosine residues ZS Zellweger syndrome xiii    LIST OF FIGURES Figure 1. 1: A single protein crystal 3 Figure 1. 2: Unit-cells and 14 Bravais lattices 4 Figure 1. 3: The two types of interference... Data Bank PEX Peroxisome PTK6 Protein tyrosine kinase 6 PTKs Protein tyrosine kinases PTS1 Peroxisome targeting signal 1 PTS2 Peroxisome targeting signal 2 RADAR The peroxisomal receptor accumulation and degradation in the absence of recycling Rak Also named GTK/Bsk/IYK xii    SAD Single wavelength anomalous dispersion SH2 Src homology 2 domain SH3 Src homology 3 domain Sik salt-inducible kinase Src Sarcoma... purification of GST-Pex8 by lysozyme addition via affinity chromatography using GSTrap FF 1 mL from GE Healthcare 57 Figure 4 .16 : Gel filtration profile for GST-Pex8 59 Figure 4 .17 : Anion exchange purification of GST-Pex20 61 Figure 4 .18 : Peptide mass fingerprinting of GST-Pex8 62 Figure 4 .19 : Peptide mass fingerprinting of GST-Pex20 62 xv    ... 2.6: Models for the role of ubiquitylation in receptor recycling (or dislocation) from the peroxisome membrane to the cytosol, and in degradation by the RADAR pathway 29 Figure 4 .1: Overexpression of Brk 43 Figure 4.2: WT-Brk was over-expressed in inclusion body after 3 hours induction 44 Figure 4.3: Purification of Brk 44 Figure 4.4: Size-exclusion chromatography profile of refolded WT-Brk 45 Figure... Frk Fyn-related kinase Gtk Gut tyrosine kinase HEK Human Embryonic Kidney IRD Infantile Refsum disease xi    Iyk Intestinal tyrosine kinase MAD Multi wavelength anomalous dispersion mPTSs Membrane peroxisome targeting signal Myr Myristoylation NALD Neonatal adrenoleukodystrophy NMR Nuclear Magnetic Resonance NOE Nucleus of normal oral epithelium OSCC Oral squamous cell carcinomas PBDs Peroxisome biogenesis...LIST OF ABBREVIATIONS AAA-ATPases ATPases Associated with various cellular Activities ABC ATP-binding cassette ALK anaplastic lymphoma kinase ATP Adenosine diphosphate BRK Breast tumor kinase Bsk β-cell Src-homology tyrosine kinase CCD Charged coupled device CTCL Cutaneous T cell lymphoma DLS Dynamic light scattering ... dispersion 10 Model building and refinement 11 1. 5.2 .1 R Factor 11 Validation and presentation 11 1. 5.3 .1 Ramachandran Plot 12 1. 5.3.2 Folding profile methods 12 CHAPTER BIOLOGY BACKGROUND 2 .1 2.2 BREAST. .. COLLECTION 1. 5 STRUCTURE DETERMINATION iv    1. 5 .1 Phasing techniques 1. 5.2 1. 5.3 1. 5 .1. 1 Direct method 1. 5 .1. 2 Molecular replacement 1. 5 .1. 3 Multiwavelengh isomorphous replacement 10 1. 5 .1. 4 Anomalous... BACKGROUND 2 .1 2.2 BREAST TUMOR KINASE (BRK) 13 2 .1. 1 Protein tyrosine kinase in signal transduction 13 2 .1. 2 Brk family non-receptor tyrosine kinases 15 2 .1. 3 Brk 16 2 .1. 4 Role of Brk in lymphoma 20