HETEROGENEITY OF PROTEIN CONFORMATION IN SOLUTION FROM THE LIFETIME OF TRYPTOPHAN PHOSPHORESCENCE

The decay of Trp phosphorescence of proteins in fluid solutions was sh own to provide a sensitive tool for probing the conformational homogen eity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential d ecays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which t he conformer equilibrium is derived, was found to be a sensitive funct ion of solvent composition (buffer, pH, ionic strength and glycerol co solvent), temperature, and complex formation with substrates and cofac tors. In many cases, raising the temperature, a point is reached at wh ich the decay becomes practically monoexponential, meaning that confor mer interconversion rates have become commensurate with the triplet li fetime. Estimation of activation free energy barriers to interconversi on shows that the large values of Delta G are rather similar among po lypeptides and that the protein substates involved are sufficiently lo ng-lived to display individual binding/catalytic properties.