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Effect of temperature on the mechanism of actin polymerization
Abstract:
The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.
Insights
Temperature significantly impacts G-actin polymerization, with dimer formation being the most sensitive step. Increasing temperature enhances G-actin monomer isomerization, affecting magnesium and calcium ion binding dynamics.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Dynamics
Background:
- Actin polymerization is crucial for cellular functions.
- Understanding the kinetics of actin polymerization is key to cellular mechanics.
Purpose of the Study:
- To investigate the temperature-dependent kinetics of Mg2+-induced G-actin polymerization.
- To elucidate the role of temperature in actin monomer dynamics and filament formation.
Main Methods:
- Kinetic analysis of G-actin polymerization at varying temperatures (10-35°C).
- Utilized Mg2+, Ca2+, and G-actin concentration variations.
- Employed N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin and reaction simulations.
- Measured elongation rates using plasma gelsolin-nucleated actin.
Main Results:
- A polymerization mechanism consistent with previous findings fit the data across the tested temperature range.
- Dimer formation exhibited the highest temperature sensitivity, with rate constant ratios changing significantly.
- Increasing temperature promoted reversible Mg2+-induced G-actin monomer isomerization.
- Mg2+ binding affinity increased, while Ca2+ binding affinity decreased with rising temperature.
Conclusions:
- Dimer formation is the rate-limiting and most temperature-sensitive step in Mg2+-induced G-actin polymerization.
- Temperature influences G-actin monomer conformation and ion binding, impacting polymerization kinetics.
- Actin filament elongation rates show a temperature dependence slightly exceeding diffusion-limited predictions.