THE USE OF A LONG-LIFETIME COMPONENT OF TRYPTOPHAN TO DETECT SLOW ORIENTATIONAL FLUCTUATIONS OF PROTEINS

The membrane protein porin and a synthetic polypeptide of 21 hydrophob ic residues were inserted into detergent micelles or lipid membranes, and the fluorescence of their single tryptophan residue was measured i n the time-resolved and polarized mode. In all cases, the tryptophan f luorescence exhibits a long-lifetime component of about 20 ns, This lo ng-lifetime component was exploited to detect slow orientational motio ns in the range of tens of nanoseconds via the anisotropy decay. For t his purpose, the analysis of the anisotropy has to be extended to acco unt for different orientations of the dipoles of the short- and long-l ifetime components. This is demonstrated for porin and the polypeptide solubilized in micelles, in which the longest relaxation time reflect s the rotational diffusion of the micelle. When the polypeptide is ins erted into lipid membranes, it forms a membrane-spanning alpha-helix, and the slowest relaxation process is interpreted as reflecting orient ational fluctuations of the helix.