Red-edge excitation study of nonexponential fluorescence decay of indole in solution and in a protein

Proteins are known to be heterogeneous systems with a hierarchy of internal motions. However, those properties are often ignored when the complex fluo rescence decay of tryptophan residues is compared to model studies with ind ole derivatives in solution. Here two simple models are presented, which il lustrate different aspects of protein organization: (1) Trp zwitterion in b uffer exemplifies ground-state heterogeneity and (2) indole in water/glycer ol mixture exemplifies excited-state reconfiguration of solvate. Both syste ms are known to produce nonexponential fluorescence decay, attributed to th e existence of multiple species (rotamers) or to the effects of slow dipola r relaxation, for (1) and (2), respectively. In the latter case a substanti al dependence of decay on the excitation wavelength is expected. Indeed suc h dependence is observed for indole in water/glycerol mixture but not for T rp zwitterion in buffer. Therefore, excitational dependence can be used as a criterion to distinguish effects of multiple conformations in the ground state from effects of excited state reactions on tryptophan decays in prote ins. The example of the bee venom peptide melittin indicates that both phen omena are important for interpretation of heterogeneity of decay, and there fore, caution should be exercised when assigning individual decay component s to conformational subspecies in proteins.