Revisitation of FRET methods to measure intraprotein distances in Human Serum Albumin

We revisited the FRET methods to measure the intraprotein distance between Trp-214 (used as donor) of Human Serum Albumin and its Cys-34, labelled with 1.5-Iaedans (used as acceptor). Variation of Trp fluorescence emission in terms of both intensity and lifetime, as well the enhancement of the acceptor fluorescence emission upon Trp excitation, have been monitored. A careful statistical analysis of the fluorescence results from ten independently prepared samples, combined with suitable spectral corrections, provided reproducible distances estimations by each one of the three methods. Even if monitoring of the donor lifetime variation in the presence of the acceptor reproduces at the best the crystallographic data, by allowing even sub-nanometre distance variations to be appreciated, we suggest that a comparative analysis of all the three methods, applied with statistical significance, should be preferred to achieve a better reliability of the FRET technique.