On the Interpretation of subtilisin Carlsberg Time-ResolvedFluorescence Anisotropy Decays: Modeling with Classical Simulations

In this work, we discuss the challenging time-resolved fluorescence anisotropy of subtilisin Carlsberg (SC), which contains a single Trp residue and is a model fluorescence system. Experimental decay rates and quenching data suggest that the fluorophore should be exposed to water, but the Trp is partially buried in a hydrophobic pocket in the crystallographic structure. In order to study this inconsistency, molecular dynamics simulations were performed to predict the anisotropy decay rates and emission wavelengths of the Trp. We confirmed the inconsistency of the crystallographic structure with the experimentally observed fluorescence data and performed free energy calculations to show that the buried Trp conformation is 2 orders of magnitude (∼3 kcal/mol) more stable than the solvent-exposed one. However, molecular dynamics simulations in which the Trp side chain was restricted to solvent-exposed conformations displayed a maximum Trp emission wavelength shifted toward lower energies and decay rates compa...