Thermodynamics and Relaxation during Actin Polymerization

Actin is the most abundant cytoskeletal protein, accounting for 20% of the total protein content in all eukaryotic cells, especially muscle cells. Actin polymerizes from the globular state known as G-actin monomer to the filamentous semi-flexible polymer …


Chapter 1: Introduction and Motivation
1.1. Introduction
1.2. The Chemistry and Biochemistry of Actin
1.3. Actin Structure and Properties
1.4. The Mechanism of Actin Polymerization
1.5. Significance
1.6. Project Summary
Chapter 2: Experimental Framework
2.1. Measurement of the Extent of Polymerization under Pressure
2.2. Theory of Volume Change from Pressure Effects
Chapter 3: Sample Preparation and Experimental Methodology
3.1. Introduction
3.2. Sample Preparation
3.2.1 Preparation of Muscle Acetone Powder
3.2.2 Actin isolation and purification
3.2.3 Labeling of actin with N-(1-pyrenyl) iodoacetamide
3.2.4. Solvent exchange of H2O for D2O
3.2.5. Measuring concentration with UV/VIS spectroscopy
3.3. Fluorescence Spectroscopy: Pressure Effects and Kinetics
3.3.1 Introduction
3.3.2 Description of the Spectrofluorimeter and Related Apparatus
3.3.3 Procedure for Measuring φ (T) and Kinetics
Chapter 4: Results/Discussion of Actin Polymerization at 0.1 MPa in H2O Buffer
4.1. Run #1: Solvent=H2O, P=0.1 MPa, [Actin]=3.1 mg/mL
4.1.1 Extent of Polymerization Results
4.1.2 Initial Rates of Polymerization
4.1.3 Discussion – Thermodynamics
4.1.4 Discussion – Kinetics
4.1.5 Effect of Temperature Reversal
Chapter 5: Results/Discussion of Actin Polymerization at 0.1 MPa in D2O Buffer
5.1. Run #2: Solvent=D2O, P=0.1 MPa, [Actin]=2.93 mg/mL
5.1.1 Extent of Polymerization Results
5.1.2 Initial Rates of Polymerization
5.1.3 Discussion – Thermodynamics
5.1.4 Discussion – Kinetics
5.1.5 Effect of Temperature Reversal
5.2. Run #3: Solvent=D2O, P=0.1 MPa, [Actin]=1.0 mg/mL
5.2.1 Extent of Polymerization Results
5.2.2 Initial Rates of Polymerization
5.2.3 Discussion – Thermodynamics
5.2.4 Discussion – Kinetics
5.2.5 Effect of Temperature Reversal
5.3. The Solvent and Concentration Effect
Chapter 6: Results/Discussion of Actin Polymerization under High Pressure
6.1. Run #4: Solvent=H2O, P=10 MPa, [Actin]=3.0 mg/mL
6.1.1 Extent of Polymerization Results
6.1.2 Initial Rates of Polymerization
6.1.3 Discussion – Thermodynamics
6.1.4 Discussion – Kinetics
6.2. Run #5: Solvent=H2O, P=20 MPa, [Actin]=3.0 mg/mL
6.2.1 Extent of Polymerization Results
6.2.2 Initial Rates of Polymerization
6.2.3 Discussion – Thermodynamics
6.2.4 Discussion – Kinetics
6.3. The Volume Change of Polymerization
Chapter 7: Summary and Further Work
7.1. The Re-entrant Phase Transition
7.2. The Pressure Effect
7.3. The Volume Change
7.4. The Effect of Solvent and Hydrogen Bonding
7.5. The Relationship between rp(T) and φ(T)
7.6. The Effect of Thermal Cycling
7.7. Summary and Further Work
Appendix. Tables of the extent of polymerization as a function of time – φ(t)

Author: Matthews, Jermey Norman Augustus

Source: University of Maryland

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