[18] Studies of molecular motors using caged compounds

Publisher Summary This chapter summarizes methods developed to study cell motility using photolysis of photolabile precursors of nucleotides, nucleotide analogs, and Ca 2+ . The chapter describes preparations of motor proteins, apparatus, appropriate solutions, reagents, caged compounds, photolysis light sources, special precautions required for photolysis experiments on motor proteins, optimization of the caged molecule concentration and photolysis chamber dimensions, binding of Ca 2+ and Mg 2+ to caged ATP, and inhibition of sliding velocity of myosin and kinesin by caged ATP. Photolysis of caged molecules, particularly caged ATP and caged Ca 2+ , provides a very flexible experimental method to activate muscle fibers and motor proteins in vitro. Hence many protocols for laser pulse photolysis with these proteins have been described. Combination of photolysis with other biophysical signals that probe molecular events is a fertile area for investigation. Many of these considerations should be applicable to other macromolecular systems.