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Kinetic characterization of myosin head fragments with long-lived myosin.ATP states
A L Friedman1, M A Geeves, D J Manstein
1Department of Biochemistry, Stanford University, California 94305, USA.
Abstract:
We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.
Insights
Dictyostelium myosin II mutations E459V and E476K dramatically slow ATP hydrolysis, creating long-lived myosin.ATP states. These myosin constructs are valuable tools for studying the myosin ATPase cycle and actin interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Myosin II is a motor protein crucial for cellular processes.
- Understanding the myosin ATPase cycle is key to deciphering its mechanical functions.
- Point mutations can alter myosin's enzymatic and kinetic properties.
Purpose of the Study:
- To characterize the ATPase cycles of Dictyostelium myosin II nonhydrolyzer point mutations E459V and E476K.
- To investigate the impact of these mutations on ATP hydrolysis and actin binding.
- To evaluate the utility of these mutant constructs as tools for studying myosin function.
Main Methods:
- Expression of soluble myosin head fragment M761-1R with E459V and E476K mutations.
- Transient kinetic analyses to characterize ATPase cycles.
- Measurement of ATP off-rate constants and actin-binding affinities.
Main Results:
- Mutations E459V and E476K significantly reduce the rate of ATP hydrolysis (by 4-6 orders of magnitude).
- Both mutants form long-lived myosin.ATP states.
- E459V allowed direct measurement of ATP off-rate (2 x 10^-5 s^-1), accelerated by actin.
- E476K exhibits significantly weaker actin affinity compared to wild-type, while E459V interacts normally.
Conclusions:
- The E459V and E476K mutations provide valuable tools for studying the myosin ATPase cycle.
- These constructs facilitate research into how ATP binding affects actin affinity.
- Further investigation using these mutants can elucidate structural features of the myosin ATPase cycle.