The Use of FRET in the Analysis of Motor Protein Structure

Fluorescence resonance energy transfer (FRET) is a spectroscopic phenomenon that consists of long-range dipole-dipole interaction between two chromophores. This method can be employed to gain quantitative distance information on macromolecules. FRET is particularly useful to characterize structural states of motor proteins, because the spatial relationship between various mechanical elements of the motor undergoing its mechanical cycle is essential to understand how force and movement are generated. In this chapter, we describe the technique, including the equations, methods of introducing fluorescence probes in specific loci of the protein, and data analysis. Practical guidelines and hints are also provided for protein preparation, labeling, and measuring FRET efficiency. The protocol is presented for interhead distance measurements in the dimeric kinesin-like motor, Ncd. However, it can easily be adapted to many other motor proteins.