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Single-Particle Cryo-EM Study of the Ric-8A:Gαi1 Complex

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Single-Particle Cryo-EM Study of the Ric-8A:Gαi1 Complex University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2020 Single-particle Cryo-EM Study of the Ric-8A:Gαi1 Complex JAKE David JOHNSTON Mr Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation JOHNSTON, JAKE David Mr, "Single-particle Cryo-EM Study of the Ric-8A:Gαi1 Complex" (2020). Graduate Student Theses, Dissertations, & Professional Papers. 11647. https://scholarworks.umt.edu/etd/11647 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Single-Particle Cryo-EM Study of the Ric-8A:Gαi1 Complex A Thesis submitted to the University of Montana for the degree of Master’s in Biochemistry and Biophysics 2020 Jake Johnston List of Contents List of Contents ………………………………………………………………………………………ii-iii List of Tables…………………………….………………………………………………………………iv List of Figures……………………………………………………………………………………………v List of Abbreviations…………………………………………………………………..…………….vi-vii Abstract………………………………………………………………………………………………...viii Acknowledgments……………………………………………………………………………………….ix Chapter 1 Introduction…………………………………………………………………………………...1 1.1 A brief introduction to proteins………………………………………………………………………2 1.2 Introduction to cell signaling and G-Protein Coupled Receptors……………………………………2 1.3.1 The G-protein cycle in cell signaling………………………………………………………………3 1.3.2 Different Classification of heterotrimeric G-protein alpha subunits…………………………….....4 1.3.3 Lipid modifications of Heterotrimeric G-protein alpha subunits …………………….……………4 1.3.4 Structure of heterotrimeric G-protein alpha subunits………………………………………………5 1.4 Regulation of G-proteins by receptor independent GEFS…………………………………………7-9 1.5.1 Discovery of Ric-8 and its involvement in G-protein signaling………...…………....…………....9 1.5.2 Different Ric-8 isoforms interact directly with different classes of Gα subunits.…….…...…...9-10 1.5.3 Ric-8 acts as a molecular chaperone for Gα subunits in vivo…………………………………10-11 1.5.4 Ric-8 functions as a cytosolic GEF toward Gα subunits in vitro……………...………………….11 1.5.5 Casein Kinase II phosphorylation of Ric-8A enhances GEF and chaperone activity………...12-13 1.5.6 Structural and functional characterization of Ric-8A and Ric-8A:Gα……………………..…13-14 1.6 Aims and objectives of this study…………………………………………………………………..17 1.7 Applicability of cryogenic electron microscopy in protein structure determination……...…….17-18 1.7.1 A brief overview of the single-particle cryo-EM workflow………………………………………18 1.7.2 Vitrification or freezing of samples in single-particle cryo-EM………………………………18-19 1.7.3 Principles of the transmission electron microscope…………………………..……………….20-23 1.7.4 Direct electron detectors………………………………………………………………………23-24 1.7.5 Single-particle reconstruction workflow in cryo-EM…………………………………………24-25 1.7.6 CTF correction………………………………………………………………………………...25-27 1.7.7 Particle polishing in the Relion Software………………………………………………………...27 1.7.8 The use of antibodies in single-particle cryo-EM………………………………..……………27-28 Chapter 2 Materials and Methods………………………………………………………………………29 2.1.1 Media recipes……………………………………………………………………………………..30 2.1.2 List of e.coli strains……………………………………………………………………………….31 2.1.3 e.coli strain preservation………………………………………………………………………….31 2.2 Plasmid protein constructs………………………………………………………………………….32 2.2.1: Transformation of e.coli………………………………………………………………………….33 2.3: Protein purification of each component……………………………………………………………33 2.3.1: Purification of pET28a Ric-8A 1-491………………………………………………………..33-34 2.3.2: Purification of PDEST15 Ric-8A Full-length……………………………………………………35 2.3.3: Purification of PDEST15 Gαi1W258A and DeltaN31 Gia1…………………………………….36 2.3.4: Purification of myristoylated Gαi1…………………………………………………………........37 2.3.5: Nanobody purification…………………………………………………………………………...38 2.4.1: Ric-8A phosphorylation by casein kinase 2………………………………………………….…..40 2.4.2: Purification of Ric-8A:Gα:nb complexes…………………….…………………..………..…….41 2.5: SDS-PAGE…………………………………………………………………………………………41 ii 2.6: Vitrification of Ric-8A:Gα:nb complexes…………………………………………………...……..42 2.7: Operation of Talos Arctica and Titan Krios microscopes………………………………………42-47 2.8: Data processing…………………………………………………………………………………….47 2.8.1: CTF Correction…………………………………………………………………………………..47 2.8.2: Particle picking……………………………………………………………………………….47-48 2.8.3: 2D Class averaging………………………………………………………………………………48 2.8.4: Generating Initial models………………………………………………………………………...48 2.8.5: 3D classification and model refinement……………………………………………………........48 Chapter 3: Optimization of Ric-8A:Gα complexes for cryo-EM………………………………………49 3.1: Chapter Introduction……………………………………………………………………………….50 3.2: Purification of Ric-8A:Gαi1:nb complexes for cryo-EM analysis………………………………...50 3.3: Cryo-EM analysis of Phosphorylated Full-Length Ric-8A bound to mGαi1 and Nb 9156………..50 3.3.1: Micrograph inspection………………………………………………………………………..50-52 3.3.2: Particle picking, reference free 2D classification, and 3D classification………..……………….53 3.4: Cryo-EM analysis of phosphorylated Ric-8A-491:DeltaN31Gαi1:8117:8119 Complex….………53 3.4.1: Micrograph Analysis………………………………………………………………………….53-55 3.4.2: Reference free 2D classification and 3D classification………………………………………55-59 3.5: Cryo-EM analysis of phosphorylated Ric-8A-491:DeltaN31Gαi1:8431:9156 Complex……….…59 3.5.1: Micrograph Analysis……………………………………………………………………………..59 3.5.2: Reference free 2D class averages and 3D classifications……………….……………………59-63 3.6: Cryo-Em analysis of phosphorylated Ric-8A-491:DeltaN31Gαi1:8117:8109:8119………………63 3.6.1: Initial screening of pR491:DeltaN31Gαi1:3nbs on the Talos Arctica………………….………..63 3.6.2: Data collection and processing of pR491:DeltaN31Gαi1:3nbs……………………….……...63-65 3.7: Cryo-EM analysis of pR491:DeltaN31Gαi1:8117:8109:8119:9156 complex…………………….65 3.7.1: Screening of the pR491:DeltaN31Gαi1:8117:8119:8109:9156 complex………………………..66 3.7.2: Data collection and processing of the pR491:DeltaN31Gαi1:8117:8109:8119:9156 complex….66 3.7.3: Chapter summary and conclusions………………………………………………………………73 Chapter 4: Structure of the Ric-8A:Gα complex……………………………………………………….74 4.1: Chapter Introduction……………………………………………………………………………….75 4.2: Overall architecture of the cryo-EM Ric-8A:Gα structures……………………………………….75 4.3: Ric-8A interactions with Gα……………………………………………………………………75-78 4.4: Ric-8A induces conformational changes of Gα that facilitate GDP release….……………………78 4.5: Gα C-terminus binds to the Ric-8A ARM repeat trough…………………………………………..78 4.6: The role of Ric-8A phosphorylation with Ric-8A:Gα complexes……………………………........80 4.7: Ric-8A interacts with the Gα switch II and α3 regions…………………………………………….80 4.8: Ric-8A induces a conformational of the Gα helical domain…………………………………...80-81 4.9.1: Gα subtype selectivity by Ric-8A and Ric-8B…………………………………………….……..81 4.9.2: Ric-8A primes Gα for GTP binding…………………………………………………….………..82 4.9.3: Conclusions and future directions……………………………………………………………82-83 References…………………………………………………………………………………………...84-93 iii List of Tables Table 2.1.1 Media Recipes….………………………………………………………………………….30 Table 2.1.2 e.coli strains………………….…………………………………………………………….31 Table 2.1.3 TfbI and TfbII recipes for chemical transformation of e.coli…...………………………....32 Table 2.2 Lists of Plasmid protein expression constructs……………………………………..………..33 Table 2.3.1 Ric-8A 1-491 purification buffers…………………………………………...…….………34 Table 2.3.2 Ric-8A Full-length purification buffers……………………………………………..……..35 Table 2.3.3 PDEST15 Gαi1W258A and DeltaN31 Gαi1 purification buffers..,……………….………36 Table 2.3.4 mGαi1 purification buffers……………………………………………………………..37-38 Table 2.3.5 Nanobody purification buffers…………………………………………………………..…39 Table 2.3.6 Nanobodies purified for this study…………………………………………………….…..39 Table 2.4 Buffers for Phosphorylating Ric-8A by CK2…………………………………………….….40 Table 2.5.1 SDS-PAGE Running Buffer Recipe/staining and destaining recipe…………………...41-42 iv List of Figures Figure 1.1 Overview of the G-protein cycle………………………………………………………….....3 Figure 1.2 Crystal structure of Gαi1 bound to GTP…………………………………………………….6 Figure 1.3 G-domain interactions in Gαi1………………………………………………………………7 Figure 1.4 Reaction Scheme for cytosolic GEFS……………………………………………………….9 Figure 1.5 Potential mechanism of Ric-8 chaperone activity and GEF activity……………………….12 Figure 1.6 Structures of Ric-8A 1-452 and Ric8A:C-terminal peptide of Gα…………………………15 Figure 1.7.1 Single-particle cryo-EM workflow……………………………………………………….16 Figure 1.7.2 Diagram of electron microscope components……….…………………………………...21 Figure 1.7.3 Single-particle processing workflow……………………………………………………..26 Figure 3.1 Chromatogram and SDS-PAGE of Ric-8A complex purification….…...………………….51 Figure 3.2.1 Micrograph and 2D class averages from Ric-8A:mGαi1:Nb 9156 complex…………….52 Figure 3.2.2 3D map generated from Full-length Ric-8A bound to mGαi1 and Nb 9156……………..54 Figure 3.3.1 Micrographs of the phosphorylated Ric-8A-491:DeltaN31Gαi1:8117:8109 complex…..56 Figure 3.3.2 2D class averages for the pRic-8A-491:DeltaN31Gαi1:8117:8109 complex……….…...57 Figure 3.3.3 3D map generated for the pRic-8A-491:DeltaN31Gαi1:8117:8109 complex…………...58 Figure 3.4.1 Micrograph and 2D class averages of pRic-8A-491:DeltaN31Gαi1:8431:9156 CHAPS..60
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