Molecular motors play a vital role in wide range of biological processes in addition to generation of movement by muscles. The kinesin superfamily of motors moves a variety of cargoes along microtubules in cells. Although much is known about how these motors couple ATP hydrolysis to generation of motility, there are still significant gaps in our understanding of the energetics of the process. Work will be conducted to determine the energy change for each of the major steps in ATP hydrolysis for both the microtubule stimulated pathway and the microtubule independent pathway. This analysis will provide an integrated view of the complete energetic cycle of kinesin when moving along microtubules. Extensive use will be made of the powerful technique of 18-O oxygen exchange reactions. This technique provides important information about the reversibility of the reactions that is difficult or impossible to obtain by other means. Although the forward rates in the hydrolysis direction are fairly well understood, determination of the free energy change at each step requires evaluation of the equilibrium constant which means having to know the rate of the reverse reaction. Extensive use will also be made of Dr. Hackney's recent discovery that an N-terminal domain (Nte) of the kinesin family member BimC functions to tether the motor to MTs without significant perturbation of the kinetics of either ATP hydrolysis or motility. This will allow studies to be performed at saturating concentrations of MTs that were not previously possible. Stopped flow fluorescence techniques using mant-nucleotides will be an additional important approach. This kinetic analysis will also be extended to other superfamily members as appropriate, particularly as many are slower than kinesin-1 and can be more readily analyzed.
This project has broader implications for the nature of energy coupling between different biological processes. It is also important for extending our fundamental understanding of how biological motors work and how they may be modified. This information will be of particular use in the design of novel motors with unique properties. Graduate education will be a central component and this research will constitute the thesis project of a graduate student. This will provide critical training as well as advance scientific knowledge. In addition, the project will heavily involve undergraduates in research roles. Previous work from Dr. Hackney's laboratory has involved over 40 undergraduates in research with several of them making sufficient contributions to be coauthors on publications. Dr. Hackney is currently the director of the Summer Undergraduate Research Program of the Department of Biological Sciences at Carnegie Mellon University. Participants in this program will be heavily involved in the work on motor proteins.
