How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?

Tryptophan fluorescence is a powerful tool for studying protein structure a nd function, especially membrane-active proteins and peptides, It is arguab ly the most frequently used tool for examining the interactions of proteins and peptides with vesicular unilamellar model membranes. However, high lig ht scattering associated with vesicular membrane systems presents special c hallenges. Because of their reduced light scattering compared to large unil amellar vesicles (LUV), small unilamellar vesicles (SW) produced by sonicat ion are widely used membrane models. Unfortunately, SW, unlike LW, are meta stable and consequently unsuitable for equilibrium thermodynamic measuremen ts. We present simple and easily implemented experimental procedures for th e accurate determination of tryptophan (Trp) fluorescence in either LUV or SW, Specifically, we show that Trp spectra can be obtained in the presence of up to 6 mM LUV that are virtually identical to spectra obtained in buffe r alone, which obviates the use of SW, We show how the widths and peak posi tions of such spectra can be used to evaluate the heterogeneity of the memb rane conformation and penetration of peptides, Finally, we show how to use a reference fluorophore for the correction of intensity measurements so tha t the energetics of peptide partitioning into membranes can be accurately d etermined. (C) 2000 Academic Press.