| Actin-Myosin Interactions | |
| An Overview of the Actin-Myosin Interactions | |
| Structural Changes in Actin and Myosin Due to Their Strong and Weak Interactions | p. 2 |
| Fluorescence Resonance Energy Transfer in Actomyosin Complexes | p. 2 |
| Insights into Actomyosin Interactions from Actin Mutations | p. 2 |
| Role of Charges in the Actomyosin Complex | p. 3 |
| The Alanine-Scanning Mutagenesis of Dictyostelium Myosin II at the Ionic Interface with Actin | p. 4 |
| Familial Hypertrophic Cardiomyopathic Mutations That Affect the Actin-Myosin Interaction | p. 4 |
| Coupling Between Chemical and Mechanical Events and Conformation of Single Protein Molecules | p. 4 |
| References | p. 5 |
| Changes in Actin and Myosin Structural Dynamics Due to Their Weak and Strong Interactions | |
| Introduction | p. 7 |
| Changes in Myosin Structural Dynamics Induced by Actin | p. 8 |
| Global Motions of Catalytic Domain: Disorder-to-Order Transition | p. 10 |
| Global Motion of Light Chain Domain: Disorder-to-Order Transition, Two Angles | p. 11 |
| Internal Motion of the Myosin Catalytic Domain: Resolve Three Structural States | p. 12 |
| Changes in Actin Structural Dynamics Induced by Myosin | p. 12 |
| Global Dynamics of Actin | p. 12 |
| Internal Dynamics ofActin | p. 14 |
| Summary and Conclusions | p. 15 |
| References | p. 17 |
| Fluorescence Resonance Energy Transfer in Acto-Myosin Complexes | |
| Structure and Function ofthe Acto-Myosin Complex | p. 21 |
| Fluorescence Resonance Energy Transfer | p. 23 |
| Results and Discussion | p. 25 |
| References | p. 29 |
| Insights into Actomyosin Interactions from Actin Mutations | |
| Introduction | p. 31 |
| Use of Actin Mutants in Actomyosin Studies | p. 33 |
| D24/D25 and E99/E100 in Loops 18-29 and 93-102 | p. 34 |
| Acidic N-Terminus 2-5 | p. 37 |
| Specificity of Actomyosin Weak Binding | p. 41 |
| Hydrophobic Strong Binding Residues | p. 42 |
| C-Terminus of Actin and the 262-274 Plug | p. 44 |
| Conclusions | p. 45 |
| References | p. 47 |
| Role of Charges in Actomyosin Interactions | |
| Introduction | p. 51 |
| Structure ofthe Actomyosin Interface | p. 51 |
| Dynamics of the Actomyosin Complex | p. 53 |
| Role ofthe Ionic Interactions | p. 53 |
| Studies of the Ionic Interactions by Chemical Cross-Linking Experiments | p. 54 |
| Cross-Linking Reactions and Identification of the Cross-Linking Sites | p. 55 |
| Regulation of the Cross-Linking Sites by Nucleotide Analogues | p. 56 |
| A New Model for the Actomyosin Interface During the ATPase Cycle | p. 58 |
| Conclusions | p. 60 |
| References | p. 61 |
| The Alanine-Scanning Mutagenesis of Dictyostelium Myosin II at the Ionic Interface with Actin | |
| Introduction | p. 65 |
| Materials and Methods | p. 67 |
| Construction and Expression of Recombinant Myosins | p. 67 |
| Phenotypes of the Transformed Cells | p. 67 |
| Myosin Purification | p. 67 |
| ATPase Assays | p. 68 |
| In Vitro Motility Assays | p. 68 |
| Results and Discussion | p. 68 |
| Phenotypes of Dictyostelium Cells Expressing the Mutants | p. 68 |
| Characterization of Purified Mutant Myosins | p. 69 |
| References | p. 73 |
| Familial Hypertrophic Cardiomyopathic Myosin Mutations That Affect the Actin-Myosin Interaction | |
| Description of the FHC Disease | p. 75 |
| Myosin FHC Mutations | p. 76 |
| Myosin FHC Mutations That Affect the Actin-Myosin Interaction | p. 77 |
| Myosin FHC Mutations Near the Actin-Binding Interface | p. 77 |
| Myosin FHC Mutations Near the ATP Binding Site | p. 80 |
| Light Chain FHC Mutations That Affect the Actin-Myosin Interaction | p. 80 |
| Actin Mutations That Cause FHC | p. 81 |
| Mechanism for the FHC Disease | p. 82 |
| Conclusions | p. 83 |
| References | p. 83 |
| Coupling between Chemical and Mechanical Events and Conformation of Single Protein Molecules | |
| Introduction | p. 87 |
| Measurements of Force Exerted by Single Myosin Molecules | p. 88 |
| Visualization of Turnover of Single ATP Molecule | p. 90 |
| Coupling Between Chemical and Mechanical Events | p. 93 |
| Detection of Conformation of Single Protein Molecules | p. 98 |
| Fluorescence Resonance Energy Transfer of a Single Protein Molecule | p. 98 |
| Conformational Diversity of Myosin Subfragments | p. 100 |
| Dynamic Behavior of Protein Molecules | p. 102 |
| Concluding Remarks | p. 103 |
| References | p. 104 |
| Actin-Based Calcium Regulation | |
| An Overview of Actin-Based Calcium Regulation | |
| Introduction | p. 107 |
| Cooperativity in the Ca2+ Regulation of Muscle Contraction | p. 107 |
| Motility Assays of Calcium Regulation of Actin Filaments | p. 108 |
| The Ultrastructural Basis of Actin Filament Regulation | p. 108 |
| The Role of Troponin in the Ca2+ Regulation of Skeletal Muscle Contraction | p. 108 |
| Structural Changes Between Regulatory Proteins and Actin: A Regulation Model by Tropomyosin-Troponin Based on FRET Measurements | p. 109 |
| Cooperativity in the Ca2+ Regulation of Muscle Contraction | |
| Introduction | p. 111 |
| The Problem: The Force/pCa2+ Curve | p. 111 |
| Components of the Regulatory System | p. 112 |
| The Three Thin Filament States from Solution Studies | p. 114 |
| Actin-S1 ATPase: Two Activity States | p. 114 |
| Equilibrium Binding of S1 to Actin Filaments | p. 115 |
| Kinetics of S1 Binding to Actin Filaments: Three S1-Binding States | p. 117 |
| Relationship of Structural States to Solution States | p. 120 |
| Observation of States | p. 120 |
| Probes of Equilibrium Titrations and Kinetics | p. 120 |
| X-Ray Diffraction | p. 121 |
| Electron Microscopy | p. 121 |
| Size of the Cooperative Units | p. 122 |
| Cooperativity of Ca2+ Binding to Troponin | p. 122 |
| Cooperativity of Blocked to Closed Transition | p. 123 |
| Cooperativity of Closed to Open Transition | p. 125 |
| Relationship of Solution States to Fibers | p. 126 |
| References | p. 129 |
| Motility Assays of Calcium Regulation of Actin Filaments | |
| Introduction | p. 133 |
| Materials and Methods | p. 135 |
| Results | p. 137 |
| Discussion | p. 139 |
| Conclusion | p. 145 |
| References | p. 146 |
| The Ultrastructural Basis of Actin Filament Regulation | |
| Introduction | p. 149 |
| Regulation in Striated Muscle | p. 150 |
| Background | p. 150 |
| The "Steric Blocking" Model of Regulation | p. 152 |
| Early Controversies Concerning the Steric Model | p. 153 |
| Recent Advances on the Ultrastructural Basis of Regulation | p. 155 |
| Towards an Atomic Level Model for Regulation | p. 157 |
| Recent Criticism of the Steric Blocking Model | p. 159 |
| Mechanistic Insights into Regulation Using Mutant Tropomyosin | p. 161 |
| The Structure of Thin Filaments During Active Sliding with Myosin Filaments | p. 161 |
| Regulation in Smooth Muscle | p. 162 |
| Caldesmon | p. 163 |
| Calponin | p. 163 |
| Conclusions | p. 164 |
| Future Directions | p. 165 |
| References | p. 165 |
| The Role of Troponin in the Ca2+-Regulation of Skeletal Muscle Contraction | |
| The Role of TnC and Its Interaction with TnI in the Regulation of Contraction | p. 171 |
| The Role of TnT and Its Interaction with TnC in the Regulation of Contraction | p. 176 |
| Model of the Regulation of Contraction by Troponin | p. 183 |
| References | p. 185 |
| Structural Changes Between Regulatory Proteins and Actin: A Regulation Model by Tropomyosin-Troponin Based on FRET Measurements | |
| Introduction | p. 191 |
| History ofCa2+ Regulation | p. 191 |
| FRET as an Optical Ruler | p. 192 |
| Probe Sites on Proteins | p. 192 |
| Structural Changes Measured by FRET | p. 193 |
| Tropomyosin-Actin | p. 193 |
| Troponin-Actin | p. 196 |
| A New Model For Regulation | p. 197 |
| FRET Data Suggest a New Regulation Model | p. 197 |
| Evaluation of the Regulation Model | p. 198 |
| Conclusions | p. 200 |
| References | p. 201 |
| Subject Index | p. 205 |
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