Tryptophan phosphorescence of ribonuclease T1 as a probe of protein flexibility.

The phosphorescence properties of Trp-59 of ribonuclease T1 fromAspergillus oryzae were monitored as a function of temperature, pH, salt concentration, and complex formation with substrate analogues and, also, in the presence of glycerol as viscogenic cosolvent. The results establish a rough correlation between the internal flexibility of the macromolecule, as derived from the triplet lifetime, and its stability (ΔG orT m ) toward unfolding. Below 10°C or in 70% glycerol the triplet probe distinguishes at least two gross conformations for the protein, which are characterized by a large difference in phosphorescence lifetime. It is pointed out that such structural heterogeneity does not correspond with the heterogeneity inferred from fluorescence decays and acrylamide quenching rates. Further, implications of the phosphorescence data with regard to the interpretation of acrylamide quenching of fluorescence are discussed.