Use of pyrene-labelled actin to probe actin-myosin interactions: kinetic and equilibrium studies

Studying the dynamics of the interaction between actin and myosin and how this is modulated by ATP and other nucleotides is fundamental to any understanding of myosin motor protein activity. The fluorescent label pyrene, covalently attached to actin (at Cys 374), has been one of the most useful optical probes to report myosin binding to actin. The unique spectral features of pyrene make it sensitive to changes in the microenvironment of the probe and allow to monitor processes such as conformational changes and protein-protein interactions. Here we describe how to make and use pyrene-labelled actin and describe a set of fluorescence stopped-flow measurements that allow the actin-myosin interaction to be explored at protein concentrations from μM to nM for many of the known myosin motors.