Tryptophan conformations associated with partial unfolding in ribonuclease T1.

The origin of the biexponential fluorescence decay of Trp in ribonuclease T1 under mildly destabilizing conditions, such as increased pH or temperature, or the presence of detergent, is still not understood. We have performed two extended replica-exchange molecular dynamics simulations to obtain a detailed representation of the native state at two protonation states corresponding to a high and low pH. At high pH, the appearance of partially unfolded states is evident. We found that this pH-induced destabilization originates from increased global repulsion as well as reduced local favorable electrostatic interactions and reduced H-bonding strength of His(27), His(40), and His(92). At high pH, alternative tryptophan rotamers appear and are linked to a distorted environment of the tryptophan, which also acts as a separate source of ground-state heterogeneity. The total population of these alternative conformations agrees well with the amplitude of the experimentally observed secondary fluorescence lifetime.