Biomotors and their Nanobiotechnology Applications

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