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Structural Investigation of the Molecular Chaperonin Tf55 From University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2014-01-01 Structural Investigation Of The olecM ular Chaperonin Tf55 From Thermophilic Archaeon Sulfolobus Solfataricus Sanjay Kumar Molugu University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Chemistry Commons Recommended Citation Molugu, Sanjay Kumar, "Structural Investigation Of The oM lecular Chaperonin Tf55 From Thermophilic Archaeon Sulfolobus Solfataricus" (2014). Open Access Theses & Dissertations. 1302. https://digitalcommons.utep.edu/open_etd/1302 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. STRUCTURAL INVESTIGATION OF THE MOLECULAR CHAPERONIN TF55 FROM THE THERMOPHILIC ARCHEON SULFOLOBUS SOLFATARICUS SANJAY KUMAR MOLUGU Department of Chemistry APPROVED : __________________________ Ricardo Bernal Ph.D., Chair __________________________ Mahesh Narayan Ph.D. __________________________ Siddhartha Das Ph.D. __________________________ Francia Giulio Ph.D. ______________________________________ Charles Ambler, Ph.D. Dean of the Graduate School Copyright © By Sanjay Kumar Molugu 2014 For Sulochana and Krishna Moorthy STRUCTURAL INVESTIGATION OF THE MOLECULAR CHAPERONIN TF55 FROM THE THERMOPHILIC ARCHEON SULFOLOBUS SOLFATARICUS By SANJAY KUMAR MOLUGU THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE Department of Chemistry THE UNIVERSITY OF TEXAS AT EL PASO December 2014 Acknowledgements Foremost, I would like to express my sincere gratitude to my advisor Dr. Ricardo Bernal for giving me a chance to work in his lab and for the continuous support throughout my Master’s study. Dr. Bernal’s motivation, enthusiasm and immense knowledge has helped me in all the time of research and made my research productive. I would like to thank my committee members Dr. Mahesh Narayan, Dr. Siddhartha Das, Dr. Francia Giulio for their valuable comments and suggestions. Furthermore I would also like to thank Dr. Sudheer Molugu for the cryo-EM data collection. I like to thank my fellow lab members for their help and valuable discussions. I would also like to acknowledge UTEP and Welch Foundation for the financial support for my research. v Abstract Chaperonins are ubiquitous multi-subunit macromolecules that mediate the folding and assembly of certain proteins that cannot fold properly in vivo . The Archaea bacterium Sulfolobus solfataricus encodes for three chaperonin complexes formed from three different, but closely related subunits named α, β and γ. These subunits form a homo-18mer composed of all β subunits (TF55 β) at temperatures ranging between 80 oC-85 oC, a hetero-16mer composed of α and β subunits (TF55 αβ ) at temperatures ranging between 70 oC-75 oC and a hetero 18mer composed of α, β and γ subunits (TF55 αβγ ) at temperatures ≤ 60 oC. Structures exist for the chaperonin TF55 all-β complex and for the TF55 αβ complex but not for the TF55 αβγ complex. Here we present the structural investigation of the chaperonin TF55 αβγ in the presence of ATP. The cryo-EM reconstruction of TF55 αβγ chaperonin complex revealed a double-ring of 18 subunits with nine subunits in each ring. Each of the α, β, γ subunits are arranged in an alternating fashion where three hetero-trimers reside in each ring and the intact 18- meric complex is formed by two of these rings stacked back to back, similar to other chaperonin complexes. vi Table of Contents Acknowledgements ........................................................................................... v Abstract .............................................................................................................vi Table of Contents ............................................................................................. vii List of Figures ....................................................................................................ix List of Abbreviations and Notations. .................................................................. x Chapter 1 .............................................................................................................. 1 1.1 Introduction ................................................................................................. 1 1.2 Protein folding in-vitro versus in-vivo ........................................................... 1 1.3 Molecular Chaperones and Chaperonins .................................................... 2 1.4 The Chaperonin Architecture ...................................................................... 3 1.5 Group I chaperonins .................................................................................... 4 1.6 Group II Chaperonins from Eukarya and Archaea....................................... 8 1.7 TF55 chaperonin from Sulfolobus solfataricus overview ........................... 13 Chapter 2 ............................................................................................................ 15 Biochemical Methods ...................................................................................... 15 2.1 Bacterial cell Transformation ..................................................................... 15 2.2 Bacterial cell culture and protein expression ............................................. 16 2.3 Protein purification by column chromatography ........................................ 17 2.4 Electrophoresis and SDS PAGE ............................................................... 20 vii Chapter 3 ............................................................................................................ 21 Cryo-EM overview and Single particle reconstruction technique ..................... 21 3.1 Cryo-Electron Microscope (Cryo-EM) overview......................................... 21 3.2 Negative Staining ...................................................................................... 21 3.3 Cryo-Freezing............................................................................................ 22 3.4 Single particle cryo-EM reconstruction technique...................................... 23 3.5 Three Dimensional reconstruction using EMAN ........................................ 24 3.6 Contrast Transfer function (CTF) Correction ............................................. 24 Chapter4 ............................................................................................................. 27 Structure Determination of TF55-ATP Chaperonin Complex .............................. 27 4.1 Introduction ............................................................................................... 27 4.2 Materials and Methods .............................................................................. 28 4.2.3 TF55 αβγ Complex formation .................................................................. 30 4.2.4 Cryo-electron microscopy ....................................................................... 30 4.3 Results and Discussion ............................................................................. 31 Conclusions ..................................................................................................... 38 References ...................................................................................................... 39 Curriculum Vitae .............................................................................................. 46 viii List of Figures Figure 1.1 Architecture of group I and II chaperonins ..................................................... 4 Figure1.2 Structure of the GroEL–GroES. ....................................................................... 5 Figure 1.3 The folding cycle of the bacterial chaperonin system GroEL-ES .................... 6 Figure1.4: Proposed One-Stroke Mechanism of Mt-cpn60 ............................................. 8 Figure1.5 Current understanding of the group II chaperonin mechanism...................... 10 Figure1.6 The eukaryotic TriC/CCT chaperonin. ........................................................... 11 Figure1.7 Thermosome from archaebacteria. ............................................................... 12 Figure1.8 mm-cpn from Methanococcus maripaludis. ................................................... 13 Figure1.9 Temperature dependent regulation of all the three subunits ......................... 14 Figure 2.1 General steps involved in Transformation of bacterial cells ......................... 16 Figure 2.2 General Mechanism involved in Ion Exchange Chromatography ................. 17 Figure 2.3 General Mechanism involved in Gel Filtration .............................................. 18 Figure 2.4: General Mechanism involved in Affinity Chromatography ........................... 19 Figure 2.5: General Steps involved in SDS-PAGE ........................................................ 20 Figure 3.1: Schematic representation of steps involved in Cryo-EM grid preparation .. 24 Figure 3.2 Contrast Transfer Function for the Philips CM200 FEG microscope. ........... 26 Figure 4.1: SDS PAGE gels showing the recombinant TF55 α, β and γ chaperonins purified separately and the purified TF55 αβγ complex .................................................. 33 Figure 4.2 Cryo-EM data collection
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