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1806301 by LEI HUANG CLONING, EXPRESSION, PURIFICATION AND CRYSTALLIZATION OF Pf -1806301 by LEI HUANG (Under the direction of Dr. Bi-Cheng Wang) ABSTRACT Pf -1806301, a putative archaeal Lsm protein characterized in this thesis, shows sequence similarity to both archaeal Lsm and bacteria Hfq proteins. Sm/Lsm proteins are a large family of RNA-binding proteins that have been identified in archaea, bacteria and eukarya. The determination of the three-dimensional structure of Pf -1806301 therefore will not only help reveal the functions of the Lsm proteins, but also offer in-depth implications to the relationship between Lsm and Hfq proteins. In order to solve the crystal structure of Pf -1806301, the gene encoding Pf -1806301 was recloned into vector pET28, which provides a thrombin cleavage site for the removal of the His- tag after protein purification. The best Se-Met crystal diffracted up to 2.7Å, showing a significant improvement in diffraction compared with crystals grown from the protein with the His-tag attached. In addition, diffraction data reveal that the Pf -1806301 complex is of a dimer of octamers. INDEX WORDS: Pyrococcus furiosus, Archaea, Sm/Lsm, Hfq, CLONING, EXPRESSION, PURIFICATION AND CRYSTALLIZATION OF Pf -1806301 by LEI HUANG B.S., NanKai University, P.R.China, 2000 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2004 ©2004 Lei Huang All Rights Reserved CLONING, EXPRESSION, PURIFICATION AND CRYSTALLIZATION OF Pf -1806301 by Lei Huang Major Professor: Bi-Cheng Wang Committee: John P. Rose William Lanzilotta Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia Demember 2004 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my major professor Dr. Bi-Cheng Wang for his guidance, encouragement and support, which is essential for me to complete my master’s project. And I appreciate the knowledge and experience gained during the years. I would like to thanks my committee members Dr. John Rose and Dr. William Lanzilotta for their suggestions and advice on the project; Dr. Zhi-Jie Liu and Jeff Habel for their help in crystallization and data collection. I would also like to thank all my fellow members in Dr. Wang’s lab, especially Peter Horanyi and Lirong Chen. Special thanks to my husband and my parents for their constant supports and encouragements. Also thanks to all the other colleagues for their advice and supports. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS……………………………………………………………………..iv LIST OF TABLES……………………………………………………………………………. vii LIST OF FIGURES…………………………………………………………………………...viii CHAPTER 1 INTRODUCTION Hyperthermophiles and Pyrococcus furiosus ……………………………………1 Pf -1806301 is a putative Sm-like protein………………………………………. 3 Oligomerization and RNA-binding of Sm/Lsm proteins………………………...8 2 MATERIAL AND METHODS Materials………………………………………………………………………...15 Sequence Analysis…...……………………………………………….................16 Cloning and Expression…………………………………………………………16 Purification of Recombinant Pf -1806301………………………………..……..18 Thrombin Cleavage of His-tagged protein……………………………………...19 Dynamic Light Scattering ………………………………....................................20 Gel-Shift Assay……………………………….....................................................21 Expression and Purification of Se-Met protein………………………………….22 Crystallization…………………………………………………………………....22 Data Collection…………………………………………………………………..23 v 3 RESULTS Sequence Analysis……………………………………………………………….24 Cloning and Expression………………………………………………………….24 Purification of Recombinant Pf -1806301……………………………………….28 Thrombin Cleavage of His-tagged protein……………………………………....28 Dynamic Light Scattering ……………………………………………………….28 Gel-Shift Assay…………………………………………………………………..31 Expression and Purification of Se-Met protein…………………………………..31 Crystallization…………………………………………………………................34 Data Collection and Processing………………………………………………….35 4 DISCUSSION……………………………………………………………………........…..44 REFERENCES……………………………………………………………….............................47 vi LIST OF TABLES Page Table 1.1: The hyperthermophilic genera: organisms that grow optimally at 90 °C……………..2 Table 1.2: Classification of Sm/Lsm family in human, yeast, archaea and bacteria …………….6 Table 3.1: Statistics of current Sm, Lsm and Hfq crystal structures…………………………….26 Table 3.2: Data collection statistics of crystals soaked with heavy atom……………………….38 Table 3.3: Data collection parameters of P1 space group crystal………………………………..40 Table 3.4: Matthews coefficient of P1 space group crystal……………………………………...43 vii LIST OF FIGURES Page Figure 1.1: The rooted universal phylogenetic tree ………………………………………… 4 Figure 1.2: Crystal structure of Lsm1 monomer from P. abyssi (1H64)……………………..9 Figure 1.3: Sequence and structural comparison of the Sm-fold in Sm, Lsm and Hfq proteins form human, archaea and bacteria respectively………………………………………..........10 Figure 1.4: Oligomeric organizations of Sm/Lsm and Hfq complexes……………………...11 Figure 1.5: The internal uridine-binding pocket of P. abyssi Lsm1 complex (1M8V)……...14 Figure 3.1: Sequence alignment of archaeal Sm-like proteins with human canonical Sm (hsm) proteins and bacterial Hfq proteins…………………………………………………………..25 Figure 3.2: Purification scheme……………………………………………………………...27 Figure 3.3: Pf -1806301 was purified over Ni-NTA agarose………………………………..29 Figure 3.4: Superdex-75 gel filtration purification of Pf -1806301…………………………30 Figure 3.5: Dynamic light scattering analysis of native Pf -1806301 in solution……….…..32 Figure 3.6: In vitro binding of the fluorescence labelled oligo U with Pf -1806301………..33 Figure 3.7: Crystallization of Pf -1806301…………………………………………………..36 Figure 3.8: The diffraction of the best Se-Met crystal……………………………………….37 Figure 3.9: Self-rotation function plot with κ = 45°…………………………………………41 Figure 3.10: Self-rotation function plot with κ = 180°……………………………………...42 viii CHAPTER 1 INTRODUCTION Hyperthermophiles and Pyrococcus furiosus Structural genomics (1) is a multidisciplinary research project that involves bioinformatics, molecular biology, biochemistry, and biophysical methods such as NMR spectroscopy and x-ray crystallography. The objective of structural genomics is to determine the unique three-dimensional structures of proteins throughout the genome in order to better understand the relationship between protein sequence, structure and function. The Southeast Collaboratory for Structural Genomics (SECSG) is one of the original seven NIH-funded Pilot Centers for high throughput (HTP) structure determination. It consists of five collaboration institutes: The University of Georgia, the University of Alabama at Birmingham, the University of Alabama at Huntsville, Georgia State University and Duke University Medical Center (2). One of the major model organisms selected by SECSG is Pyrococcus furiosus. The Latin name Pyrococcus stands for “the fireball”, which indicates its ability to grow at temperatures above 100°C. Based on its favorable growth temperature, Pyrococcus belongs to a group of organisms called hyperthermophiles. The first hyperthermophile was isolated from shallow marine volcanic vents by Stetter and coworkers in 1982 (3). Since then, more than 20 genera of hyperthermophiles have been discovered (Table 1.1) (4a). Hyperthermophilic organisms are distributed in the geothermally-heated ecosystems. They are mostly obligate anaerobes, and they 0 depend on electron donors like elementary sulfur (S ) instead of O2 for their energy conservation (4b). 1 Table 1.1 The hyperthermophilic genera: organisms that grow optimally at 90 °C (a) (b) (c) Genus Tmax (°C) Metabolism Substrate Acceptors S0-depentdent archaea Thermofilum 100 Hetero Pep S0, H+ Staphylothermus 98 Hetero Pep S0, H+ Thermodiscus 98 Hetero Pep S0, H+ Desulfurococcus 90 Hetero Pep S0, H+ 0 + Thermoproteus 92 Hetero (auto) Pep, CBH (H2) S , H 0 + Pyrodictum 110 Hetero (auto) Pep, CBH (H2) S , H Pyrococcus 105 Hetero Pep S0, H+ Thermococcus 97 Hetero Pep, CBH S0, H+ 0 + Hyperthermus 110 Hetero Pep (H2) S , H 0 2- Stetteria 103 Hetero Pep + H2 S , S2O3 0 - Pyrobaculum 102 Hetero (auto) Pep (H2) S , mO2, NO3 0 0 Acidianus 96 Auto S , H2 S , O2 S0-indepentdent archaea Sulfobococcus 95 Hetero Pep - Aeropyrum 100 Hetero Pep O2 2- - Pyrolobus 113 Auto H2 S2O3 NO3 mO2 Sulfate-reducing archaea 2- 2- Archaeoglobus 95 Hetero (auto) CBH (H2) SO4 S2O3 Iron-oxidizing archaea 2+ - 2- - Ferroglobus 95 Auto Fe , H2, S2 S2O3 NO3 Methanogenic archaea Methanococcus 91 Auto H2 CO2 Methanothermus 97 Auto H2 CO2 Methanopyrus 110 Auto H2 CO2 Bacteria Thermotoga 90 Hetero Pep, CBH S0, H+ 0 - Aquifex 95 Auto S (H2) NO3 mO2 From Roopali, R. Ph. D dissertation, University of Georgia, 2001 (a) Maxium growth temperature (b) Heterotrophic (hetero) of autotrophic (auto) growth mode (c) Growth substrates: peptides (Pep), carbohydrates (CBH), hydrogen (H2) and elemental sulfur (S0) as electron receptor 2 The classification of hyperthermophiles offers new insight into the origin of life. Based on 16S-rRNA analyses of known hyperthermophilic microbes, except for two bacterial genera, all of the hyperthermophile genera are classified as Archaea (formerly known as Archaeabacteria) (5). In 1990, Woese et al. proposed a universal phylogenetic tree for all living organisms, in which the living world is divided into three domains: the Bacteria, the Archaea, and the Eukarya (5). The Archaea can further be divided into two kingdoms: the Crenarchaeota and the Euryarchaeota. The Crenarchaeota contains
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