The long term goal is to elucidate the general mechanism of motor protein systems by developing a detailed kinetic scheme for three systems, actomyosin-myofibrils, microtubule-kinesin and microtubule-ncd. The three systems have different requirements.Muscle systems have a high contraction velocity by additive effects of many cross bridges, an efficient conversion of ATP free energy into external work and power output depends on load. Kinesin and kinesin like proteins transport organelles within cells, including vesicle transport in axons,vesicle traffic in the ER-Golgi-plasma membrane system, movements of mitochondria ER membrane system, nuclei and probably play a role in mitosis.A low velocity is maintained against a small viscous drag. But a single motor protein is required to transport a vesicle along a microtubule without detaching. Ncd is a protein with high sequence homology to kinesin in the motor domain yet it moves in the opposite direction along microtubules. The number of steps in the mechanism and the rate and equilibrium constants of the steps and therefore the pathway of the reaction will be determined by transient kinetic analysis using stop flow and rapid quenching techniques. A detailed kinetic mechanism is available for actomyosin in solution and it will be extended to myofibrils. The validity of the scheme will be tested by prediction of the velocity of motion and energy utilization. The pathway and the step(s) in which strain force is generated in muscle has been extensively investigated and there is reasonable agreement that force is developed after the hydrolysis step. The mechanism of kinesin microtubule system will be investigated for a single head motor domain(379 amino acids) and a double headed molecule(560 amino acids) prepared from an E coli expression system. The kinetic mechanism has to account for the velocity and a pathway in which both heads are rarely dissociated.Comparison of single and double headed molecules will test for interaction between heads as a possible requirement. The step(s) in which force is developed is an open question. The ncd protein motor domain (362 amino acids) will also be purified from an E coli expression system. Comparison of the steps, the corresponding rate constants and the reaction pathway for kinesin and ncd may explain how polarity of motion might be reversed for two motors which presumably have similar structures. The study of three systems with different properties is expected to give insite into the principles of the design of molecular motors for different requirements in the cell.
