MOLTEN GLOBULE-LIKE CONFORMATION OF BARSTAR - A STUDY BY FLUORESCENCEDYNAMICS

Time-resolved fluorescence intensity and anisotropy decay measurements have been carried out on barstar, the inhibitor protein of the bacter ial ribonuclease, barnase. The intrinsic fluorescence of the three try ptophans in this protein have been used to characterize the molten glo bule-like conformation at pH 3 (A form) and the native conformation at pH 7 (N state). The fluorescence intensity decay could be fitted to a sum of two exponentials with lifetimes of 4.1 and 1.5 ns at pH 7 (N s tate) and three exponentials with lifetimes of 4.9, 1.5, and 0.2 ns Bt pH 3 (A form). The emergence of the 0.2-ns component was pH dependent with a pK of similar to 4.5. Fluorescence quenching by iodide has sho wn that the tryptophan (Trp) residues are solvent inaccessible at pH 7 and partially exposed at pH 3 (A form). Quenching by acrylamide has s uggested that the 1.5-ns decay component arises from one of the three Trp residues and the 4.1-ns component arises from the remaining two Tr p residues. Of the latter, one is buried and the other is highly acces sible to acrylamide. Decay of fluorescence anisotropy has shown that t he Trp residues are rigidly held and do not have any segmental mobilit y at pH 7. The A form is characterized by a high level of aggregation and a high degree of internal motion. The aggregated A form could be r elevant in the folding pathway of barstar when the possibility of inte raction of molten globular form with chaperone proteins is recognized. Comparison of the dynamic behavior of the Cys --> Ala mutant with tha t of the wild type ha's shown the proximity of SH group(s) to Trp resi dues.