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 fluorescence decay of tryptophan residues is compared to model studies with indole derivatives in solution. Here two simple models are presented, which illustrate different aspects of protein organization: (1) Trp zwitterion in buffer exemplifies ground-state heterogeneity and (2) indole in water/glycerol mixture exemplifies excited-state reconfiguration of solvate. Both systems are known to produce nonexponential fluorescence decay, attributed to the existence of multiple species (rotamers) or to the effects of slow dipolar relaxation, for (1) and (2), respectively. In the latter case a substantial dependence of decay on the excitation wavelength is expected. Indeed such dependence is observed for indole in water/glycerol mixture but not for Trp 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 proteins. The example of the bee venom peptide melittin indicates that both phenomena are important for interpretation of heterogeneity of decay, and therefore, caution should be exercised when assigning individual decay components to conformational subspecies in proteins.