Methods and protocols
Fluorescence for in situ structure (FISS)
Fluorescence for in situ structure (FISS) has been developed to measure the orientation of protein domains. In this technique a target protein domain is expressed in a modified form containing a suitable pair of cysteines, covalently modified with a bifunctional rhodamine, then reintroduced to its native environment. The orientation of the rhodamine dipole, and hence of the vector joining the cysteines, is measured in situ, on the millisecond time-scale, using fluorescence polarisation. The protein domain orientation is determined by multiple probes on the target domain. Performing FISS experiment involves a unique combination of expertise -- from molecular biology, protein biochemistry, muscle physiology to biophysics of data interpretation.