Biomotors and their Nanobiotechnology Applications

Description

This book – a collection of reviews and research articles by the top academics in the field – provides a glimpse of the cutting-edge technology and research being carried out and shows how researchers are utilizing this knowledge to develop new areas of study and novel applications. It serves as a valuable resource while exploring the latest advances in virus particle assembly and demonstrating how the knowledge of fundamental processes has been used to advance bio-nanotechnology. Chapters detail biophysical approaches and biomotor research, discus the latest advances in DNA/RNA nanoparticle assembly and use, and introduce the use of DNA/RNA nanoparticles for drug delivery. Cover Half Title Title Page Copyright Page Dedication Table of Contents Preface Authors Contributors Note to Reader Part I: Fundamental Mechanism of Biomotor Action Chapter 1 Biological Nanomotors with Linear, Rotation, or Revolution Motion Mechanism 1.1 Introduction 1.2 Classification of Biomotors 1.2.1 Rotation Motors 1.2.2 Revolution Motors 1.2.3 Linear Motors: Myosin, Kinesin, and Dynein 1.3 Structure of Biomotors 1.3.1 Some Motor Components Display Hexameric Arrangements 1.3.2 Motor Structural Frame 1.3.3 Channel, Pore, or Surrounding Ring 1.3.4 Factors for Distinction of Revolution Motor and Rotation Motors 1.4 Motion Mechanism 1.4.1 Energy Conversion: Transition Among Entropy, Randomness, Affinity, and Conformation Change as Driving Force 1.4.2 Mechanism of Rotation Motors 1.4.3 Mechanism of Revolution Motors 1.4.4 Mechanism of Linear Motors 1.4.5 Mechanism in Control Sequential and Coordination Among Channel Subunits 1.5 Potential Motor Applications 1.6 Concluding Remark and Perspectives Acknowledgments Competing Interests References Chapter 2 Classifications and Typical Examples of Biomotors 2.1 Typical Revolving Motors 2.1.1 DNA Packaging Motor of Double-Stranded DNA Bacteriophages 2.1.2 DNA Packaging Motor of Eukaryotic dsDNA Viruses 2.1.3 dsDNA Translocases FtsK/SpoIIIE Superfamily 2.2 Typical Rotary Motors 2.2.1 F[sub(o)]F[sub(1)] Complex 2.2.2 DNA Helicase 2.2.3 Bacterial Flagella 2.3 Typical Linear Motors References Chapter 3 Structure of Revolving Biomotors 3.1 Hexameric Arrangement of Motor Components 3.2 dsDNA Translocases of the FtsK/SpoIIIE Superfamily References Chapter 4 Structure of Rotation Motors 4.1 Structure of Flagellar Motors 4.2 Structure of F[sub(o)]F[sub(1)] ATPase References Chapter 5 Structure of Linear Motors 5.1 Structure of Myosins References Chapter 6 Mechanical Properties of Molecular Motors and the Relevance to their Biological Function 6.1 Kinesin 6.2 Myosin 6.3 F[sub(0)]F[sub(1)]-ATPase 6.4 Φ29 DNA Packaging Motor References Chapter 7 Molecular Mechanism of AAA-ATPase Motor in the 26S Proteasome 7.1 Introduction 7.2 AAA+ ATPases in Ubiquitin-Proteasome System 7.3 Conformational Changes of AAA ATPases in the 26S Proteasome 7.4 Substrate Interactions Coupled with ATP Hydrolysis 7.5 Three Modes of Coordinate ATP Hydrolysis Regulate Intermediate Functional Steps 7.5.1 Mode 1 Regulates Ubiquitin Recognition, Initial Substrate Engagement, and Deubiquitylation 7.5.2 Mode 2 Regulates CP Gating, Ubiquitin Release, and Initiation of Substrate Translocation 7.5.3 Mode 3 Regulates Processive Substrate Unfolding, Translocation, and Degradation 7.6 Evidence for a Sequential Hand-over-Hand Model 7.7 Concluding Remarks Funding Acknowledgments Conflicts of Interest References Chapter 8 General Mechanism of Biomotors 8.1 Force Generation and Energy Conversion 8.2 Motor Subunit Communication References Chapter 9 Mechanism of Revolving Motors 9.1 Revolving Motion in Biological Motors 9.2 One-Way Traffic of Revolving Biomotors References Chapter 10 Mechanism of Rotary Motors 10.1 Rotation Motion in F[sub(1)] 10.1.1 Single-Molecule Rotation Assay of F[sub(1)] 10.1.2 Torque of F[sub(1)] 10.1.3 Chemomechanical Coupling of F[sub(1)] 10.1.4 Torque Generation Steps of F[sub(1)] 10.1.5 Critical Role of Phosphate-Binding Sites in Force Generation 10.2 Rotation Motion in F[sub(o)] References Chapter 11 Mechanism of Linear Motion 11.1 Conserved Catalytic Cycle of Myosins 11.2 Nucleotide-Binding Region 11.3 Actin-Binding Region 11.4 Lever Arm Region References Chapter 12 Finding of Widespread Viral and Bacterial Revolution dsDNA Translocation Motors Distinct From Rotation Motors by Channel Chirality and Size Abbreviations 12.1 Background 12.2 Results and Discussion 12.2.1 Revolution and Rotation Motors Can Be Distinguished by Motor Channel Size 12.2.2 Conductance Assay of Single Connector Channels for Translocation of Tetra-Stranded DNA Reveals a Threefold Width of Phi29 Channels Compared to dsDNA 12.2.3 The Left-Handed Chirality of Revolution Motors is Distinct From the Right-Handed Chirality of Rotation Motors 12.2.4 Common Force Generation Mechanism of dsDNA Translocation Motors in Bacteria, Eukaryotic, and Prokaryotic Viruses 12.2.5 DNA Twists Rather Than Rotates Due to Motor Channel Conformational Changes During DNA Translocation 12.2.6 Single-Molecule Real-Time Imaging and Force Spectroscopy Revealed that No Rotation Occurs During DNA Translocation 12.3 Conclusion 12.4 Materials and Methods 12.4.1 Incorporation of the Connector Channel Into a Planar Bilayer Lipid Membrane 12.4.2 Construction of Tetra-Stranded DNA 12.4.3 Single-Channel Conduction Assays for Each Membrane-Inserted Connector Channel 12.2.4 Direct Observation of DNA Translocation Competing Interests Authors' Contributions Acknowledgments Note References Chapter 13 The ATPase of the phi29 DNA Packaging Motor is a Member of the Hexameric AAA+ Superfamily Highlights 13.1 Introduction 13.2 Results 13.2.1 Phi29 DNA Packaging Motor Contains Three Coaxial Rings 13.2.2 Native PAGE, EMSA, and CE Reveal Hexameric ATPase 13.2.3 Mutations of Known Motifs Suggest that phi29 gp16 is a Member of the AAA+ Superfamily of ATPases 13.2.4 Binomial Inhibition Functional Mutant Assays Validate Hexameric ATPase 13.3 Discussion 13.4 Materials and Methods 13.4.1 Cloning, Mutagenesis and Protein Purification 13.4.2 Measurement of gp16 ATPase Activity 13.4.3 In Vitro Virion Assembly Assay 13.4.4 Statistical Analysis and Data Plotting 13.4.5 CE Experiments to Determine Ratio of gp16 to Bound dsDNA 13.4.6 Native PAGE of eGFP-gp16 13.4.7 Atomic Force Microscopy (AFM) Imaging 13.4.8 Electrophoretic Mobility Shift Assay (EMSA) Acknowledgements References Chapter 14 Arginine Finger Serving as the Starter of Viral DNA Packaging Motors References Chapter 15 Three-Step Channel Conformational Changes Common to DNA Translocases of Bacterial Viruses T3, T4, SPP1, and phi29 15.1 Introduction 15.2 Materials and Methods 15.2.1 Materials and Reagents 15.2.2 Expression and Purification of phi29, SPP1, T3, and T4 Portals 15.2.3 Preparation of Lipid Vesicles Containing the phi29, SPP1, T4, and T3 Portals 15.2.4 Portal Insertion Into Planar Lipid Bilayer 15.2.5 Electrophysiological Measurements 15.3 Results 15.3.1 Cloning, Expression, and Purification of the Portals of phi29, SPP1, T4, and T3 15.3.2 Insertion of Portal Channels Into Lipid Membrane for Determining Channel Size Using Conductance Measurements 15.3.3 Three-Step Gating of phi29, SPP1, T4, and T3 Portal Channels 15.4 Discussion 15.5 Conclusions Author Contributions Acknowledgments References Chapter 16 Sequence Dependence of Reversible CENP-A Nucleosome Translocation 16.1 Introduction 16.2 Results and Discussion 16.3 Materials and Methods Acknowledgements References Chapter 17 Same Function From Different Structures Among Pac Site Bacteriophage (TerS) Terminase Small Subunits References Chapter 18 Kinetic Study of the Fidelity of DNA Replication with Higher-Order Terminal Effects 18.1 Introduction 18.2 Basic Theory of Steady-State Copolymerization Kinetics 18.2.1 Bernoullian Model: Zero-Order Terminal Effects 18.2.2 Terminal Model: First-Order Terminal Effects 18.2.3 Penultimate Model: Second-Order Terminal Effects 18.2.4 Higher-Order Terminal Models 18.3 DNA Replication: A Binary Copolymerization in Two Dimensions 18.3.1 Basic Theory of Steady-State Kinetics of the Exonuclease Proofreading Model 18.3.1.1 First-Order Proofreading Model 18.3.1.2 Second-Order Proofreading Model 18.3.2 The Fidelity of DNA Replication 18.3.2.1 The Infinite-State Markov Chain Method for Exonuclease Proofreading 18.3.2.2 Approximation of φ Under Bio-Relevant Conditions 18.4 Case Study: T7 DNA Polymerase 18.5 Discussion and Conclusion Acknowledgments References Chapter 19 Multilevel Control of the Activity of p97/Cdc48, A Versatile Protein Segregase Abbreviations 19.1 Diverse Cellular Functions of p97 19.1.1 Protein Quality Control and Homeostasis 19.1.2 Ribosome-Associated Quality Control 19.1.3 Chromatin-Associated Degradation 19.1.4 Mitosis and Cell Cycle 19.1.5 Membrane Fusion in Cell Division 19.1.6 Autophagy 19.1.7 Endocytosis 19.1.8 Ciliogenesis 19.2 Architecture and Molecular Characteristics of p97/Cdc48 19.2.1 Basic Architecture 19.2.2 Conformational Changes of Isolated p97 19.2.3 The Presence of Pre-bound ADP in Isolated p97 19.2.4 Asymmetry of N Domain Conformation in Wild-Type p97 19.2.5 Stair-Case Arrangement of D2 Domains of p97 in the Presence of Substrate 19.3 Observable Enzymatic Activities of p97 in Vitro 19.3.1 ATPase Activity 19.3.2 Protein Unfoldase Activity 19.3.3 Binding Affinities for Nucleotides and Adaptors/Cofactors 19.4 p97-Interacting Adaptors and Cofactors 19.4.1 Detection of p97 Interactions with Adaptors/Cofactors by Pull-Down Assay 19.4.2 Adaptors Are Used to Control Subcellular Localization and Activity of p97/Cdc48 19.4.3 Cofactors Modify Substrates for Recruitment and Release 19.5 Regulation of p97/Cdc48 Activity 19.5.1 D1 Domain has Four Different Nucleotide States 19.5.2 Mechanism of Selectivity for Adaptor Binding by p97/Cdc48 19.5.3 Communication Between Different Domains 19.6 Diseases as a Result of Altered Regulation in p97 19.7 Future Perspective Acknowledgment References Chapter 20 High-Resolution Structure of Hexameric Herpesvirus DNA Packaging Motor Elucidates Revolving Mechanism and Ends 20-Year Fervent Debate 20.1 Structural Evidence of this Report to Support the Hexamer Instead of Pentamer Structure 20.2 Structural Evidence of Conformational Change in Favor of a Revolving Over a Rotating Mechanism 20.3 Structural Evidence of Channel Size in Favor of a Revolving Over a Rotating Mechanism 20.4 Structural Evidence to Elucidate that an Arginine Finger is Involved in Controlling the Direction of Motion 20.5 Why Nature Evolved a Revolving Mechanism? 20.6 Interpretation for Why a Hexamer Motor Has Been Reported as a Pentamer Motor in Several Bacteriophage DNA Packaging Motors in History 20.7 The Broad Impact of this Work Acknowledgments References Part II: Methods for the Study of Biomotors Chapter 21 Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor 21.1 Introduction 21.2 Materials 21.2.1 Specialized Equipments 21.2.2 Buffers and Solutions 21.3 Methods 21.3.1 Methods for Single-Pore Conductance Measurements 21.3.1.1 Prepare Small Unilamellar Liposomes with Membrane-Embedded Reengineered phi29 Connectors 21.3.1.2 Set up Bilayer Lipid Membrane (BLM) Chambers and Instruments for Single-Channel Conduction Assays 21.3.1.3 Insert Connectors Into Planar Lipid Membrane and Characterize Their Conductance 21.3.2 Methods for Sensing Single DNA Molecules Using Membrane-Embedded Connectors 21.3.3 Methods for Sensing Single Chemicals or Single Antibodies Using Membrane-Embedded Connectors 21.3.3.1 Capture and Fingerprinting of Single Chemicals 21.3.3.2 Capture and Fingerprinting of Single Antibodies 21.3.4 Methods for Imaging Single RNA Nanostructures by Atomic Force Microscopy 21.3.4.1 Preparation of Mica Substrate for Immobilizing RNA Nanoparticles 21.3.4.2 AFM Imaging in Tapping Mode in Air (Figure 21.7) 21.3.5 Methods for Determining the Stoichiometry of RNA on phi29 Motor by Single-Molecule Photobleaching Assay 21.3.6 Methods for Single-Molecule Distance Measurement of RNA by FRET 21.3.7 Methods for Observing DNA Packaging by Optical Fluorescence Microscopy 21.3.7.1 Generate Biotinylated Phi29 Genomic DNA for Labeling with Fluorescent Bead 21.3.7.2 Preparation of Stalled Packaging Intermediate 21.3.7.3 Real-Time Observation of DNA Translocation with Fluorescence Microscopy 21.3.8 Methods for Observing DNA Packaging by Combining Optical Microscopy and Magnetomechanics 21.3.8.1 Preparation of Stalled Packaging Intermediate Labeled with Magnetic Beads 21.3.8.2 Real-Time Observation of DNA Translocation with Magnetomechanical System Acknowledgments Notes References Chapter 22 Instrumental Design for Five-Dimensional Single-Particle Rotational Tracking 22.1 Introduction 22.2 Results and Discussion 22.2.1 Instrumental Design of Parallax-DIC Microscopy 22.2.2 Rotational Tracking with Gold Nanorods 22.2.3 Tracking Program 22.2.4 Compatibility of 5D-SPT 22.3 Conclusions Acknowledgments References Chapter 23 The Appropriate Ratio of Retroviral Structural Proteins is Activated by the Spleen Necrosis Virus Post-Transcriptional Control Element 23.1 Introduction 23.2 Materials and Methods 23.2.1 Molecular Cloning 23.2.2 Transfections 23.2.3 Protein Analysis 23.3.4 RNA Analysis 23.3.5 SFPQ/PSF Downregulation by shRNA and Rescue by Exogenous Expression 23.3 Results 23.3.1 SNV 5'-UTR Segments Regulate the Ratio of Virion Structural Proteins 23.3.2 PCE AC' is Sufficient to Dysregulate the Ratio of SNV Unspliced and Spliced RNAs 23.3.3 Deletion of PCE and Distal 300 Increases the Stability of SNV env mRNA 23.3.4 PCE Activates Ribosome Engagement to SNV Unspliced RNA 23.3.5 SFPQ/PSF has a Vital Role in the Post-Transcriptional Expression of SNV 23.4 Discussion Acknowledgments References Part III: Application of Biomotors Chapter 24 Translation of the Long-Term Fundamental Studies on Viral DNA Packaging Motors into Nanotechnology and Nanomedicine 24.1 Introduction 24.2 Structures and Functions of the Biomotors for Translocation of Viral Genomes 24.2.1 Structure of the Viral DNA Packaging Motors 24.2.2 The Revolving Biomotors for Packaging of the Viral dsDNA Genomes 24.2.3 Translocation of dsDNA by the Substrate Revolving May Be a Common Mechanism during Biomotor Evolution 24.2.3.1 Revolving Mechanisms Are Defined by Channel Sizes of Biomotors 24.2.3.2 The Revolution Mechanisms Are Distinguished by their Chirality 24.2.3.3 Stepwise Translocation of dsDNA Results From Electrostatic Interaction 24.2.3.4 A Model has Been Proposed that the ATPase gp16 Hexamer Functions as an Open Washer Linked Into a Filament with a Left-Handed Chirality 24.2.4 Packaging of the Viral dsRNA Genomes 24.2.5 Special Aspects of the Revolving Motors 24.2.5.1 Force Generation and Energy Conversion 24.2.5.2 Unidirectional dsDNA Translocation 24.2.5.3 Communications/Interactions Between Motor Subunits for Sequential Action 24.2.5.4 The Prohead RNA Plays a Role in Motor Conformation Dynamics 24.3 The Application of the Revolving Biomotors in the Single-Pore Sensing 24.3.1 The Mechanism of the Single-Pore Sensing 24.3.2 The Connectors in the Single-Pore Sensing System 24.3.3 Application of the Biological Nanopore Sensing System in DNA, RNA, and Protein Analysis 24.4 Studies on the Bacteriophage Phi29 Motor pRNA Lead to the Emergence of RNA Nanotechnology 24.4.1 Timeline of Phi29 Motor pRNA Research in the Development of RNA Nanotechnology 24.4.2 Techniques for the Construction and the Applications of RNA Nanoparticles 24.4.2.1 Using RNA 3WJ Structure as Scaffolds 24.4.2.2 Applications of RNA Nanoparticles in RNA Interference (RNAi) Therapy 24.4.3 A Brief Summary of RNA Nanotechnology 24.5 Studies on the Poly-Homo-Subunit of the Nucleic Acid Translocation Motor Lead to the Discovery of a Method for the Development of Highly Potent Inhibitory Drugs 24.5.1 Use the Mathematical Formula of Binomial Distribution and Yanghui Triangle to Investigate the Inhibition Efficiency 24.5.2 The Nature of the Poly-Homo-Subunit of the Nucleic Acid Translocation Motor in Relation to the Drug Inhibition Efficiency 24.5.3 Extension of the Finding in the Inhibition Efficiency of Viral Motors 24.5.4 The Poly-Homo-Subunit of the Nucleic Acid Translocation Motor 24.5.5 Development of Highly Potent Drugs Against Multi-Subunit ATPases Analogous to a Series Circuit 24.6 Conclusions and Perspectives Compliance and Ethics References Chapter 25 Translocation of Peptides Through Membrane-Embedded SPP1 Motor Protein Nanopores 25.1 Results 25.1.1 Characterization of SPP1 Connector Channel Embedded Into Lipid Bilayer 25.1.2 Translocation of Peptides Through SPP1 Connector Channels and Kinetic Study 25.2 Discussion 25.3 Conclusion 25.4 Materials and Methods 25.4.1 Materials 25.4.2 Cloning and Purification of the SPP1 Connector Protein 25.4.3 Insertion of the Connector Protein Into Preformed Lipid Bilayers 25.4.4 Electrophysiological Measurements 25.4.5 Purification of the DNA/RNA Used in the Experiment 25.4.6 Translocation Experiments of DNA and RNA References Chapter 26 Insertion of Channel of phi29 DNA Packaging Motor Into Polymer Membrane for High-Throughput Sensing 26.1 Methods 26.1.1 Materials 26.1.2 Insertion of phi29 Connector Into Liposome 26.1.3 Insertion of phi29 Proteoliposome Into the Polymeric Membrane of MinION Flow Cell 26.1.4 Peptide Translocation 26.1.5 Electrophysiology Assay 26.2 Results 26.2.1 Insertion of the Channel of phi29 DNA Packaging Motor Into the Polymer Membrane 26.2.2 Confirmation of Single-Pore Insertion by the Observation of Three-Step Gating of the Channel of phi29 DNA Packaging Motor 26.2.3 Differentiation of Four Peptides Using the phi29 Motor Channel Inserted Into the Membrane of Oxford Nanopore MinION Flow Cell 26.3 Discussion Appendix A. Supplementary Data References Chapter 27 Engineering of Protein Nanopores for Sequencing, Chemical or Protein Sensing, and Disease Diagnosis 27.1 Introduction 27.2 General Strategies for Engineering Protein Nanopore or Channels 27.3 Engineering Protein Nanopores or Channels for DNA and RNA Sequencing 27.3.1 α-Hemolysin 27.3.2 phi29 and Other Channels of Viral DNA Packaging Motors 27.3.3 MspA 27.3.4 Commercial Ventures 27.4 Engineering Protein Nanopores for Single Chemical or Macro-Molecule Sensing 27.4.1 Sensing Directly Using Site-Directed Mutagenesis 27.4.2 Sensing Via Probes Introduced Through Fusion Protein Expression 27.4.3 Sensing with Non-covalent Adaptors 27.4.4 Sensing with Covalent Adaptors 27.4.5 Sensing Via Conformational Changes in the Channel 27.4.6 Changing Oligomeric State of Channel 27.5 Perspectives Acknowledgments References and Recommended Reading Chapter 28 Phage Portal Channels as Nanopore Sensors 28.1 Introduction 28.2 Membrane Integration Strategy 28.3 Sensing of Nucleic Acids by Translocation 28.4 Sensing of Peptides by Translocation 28.5 Sensing of Proteins by Capture and Fingerprinting 28.6 Sensing of Chemicals Using Probes 28.7 Perspectives and Future Outlook Conflict of Interest References Chapter 29 Controlled Co-Assembly of Viral Nanoparticles of Simian Virus 40 with Inorganic Nanoparticles: Strategies and Applications 29.1 Introduction 29.2 Co-Assembly of SV40 VNPS with Inorganic Nanoparticles to Form Hybrid Nanostructures 29.3 Encapsulation of NPS Inside SV40 VNPS for Bioimaging 29.4 Conclusions and Perspectives Acknowledgments References Chapter 30 Potential of 3Dpol as an Enzymatic Reader for Direct RNA Sequencing 30.1 Introduction 30.2 Results and Discussion 30.2.1 Hairpin-Primed RNA Synthesis 30.2.2 Initiation of RNA Synthesis 30.2.3 Hairpin Attachment to an RNA Template 30.3 Future Direction Acknowledgments References Chapter 31 Channel From Bacterial Virus T7 DNA Packaging Motor for the Differentiation of Peptides Composed of a Mixture of Acidic and Basic Amino Acids 31.1 Introduction 31.2 Materials and Methods 31.3 Results and Discussion 31.3.1 The Discrimination of Peptides with the Mixture of Positively and Negatively Charged Amino Acids 31.3.2 The Discrimination of Peptides with the Locational Difference of Single Amino Acid 31.4 Conclusions Author Contributions Conflicts of Interest Acknowledgments References Chapter 32 Nano-channels of Viral DNA Packaging Motor as Single Pore to Differentiate Peptides with Single-Amino Acid Difference 32.1 Introduction 32.2 Material and Methods 32.2.1 Materials 32.2.2 Cloning, Expression, and Purification of T7 Connector 32.2.3 Incorporation of T7 Connector Into Liposomes 32.2.4 Electrophysiological Assays 32.2.5 Peptide Translocation Assays 32.2.6 Peptide Cleavage Assay 32.3 Results and Discussion 32.3.1 Cloning and Expressing the T7 Connector in E. Coli and Insertion of the Purified Connector Into Lipid Bilayer Membrane 32.3.2 Differentiation of Peptides of Varying Residues by Current Blockage 32.3.3 Discriminating Peptides of Varying Size in Mixture 32.3.4 Mapping of 11-aa and 12-aa Peptides by Real-Time Sensing Via Trypsin Cleavage 32.4 Conclusions Author Contributions Competing Financial Interests Acknowledgements References Index