ACRYLAMIDE QUENCHING OF PROTEIN PHOSPHORESCENCE AS A MONITOR OF STRUCTURAL FLUCTUATIONS IN THE GLOBULAR FOLD

This study examines acrylamide quenching of tryptophan room-temperatur e phosphorescence in proteins and the role that factors such as long-r ange interactions and environment-dependent quenching efficiency might play in the interpretation of bimolecular quenching rate constants in terms of hindered quencher migration through the globular fold. The d istance dependence of the through-space quenching rate is evaluated by studying the effects of acrylamide on the phosphorescence intensity a nd decay kinetics-of the indole analogue 2-(3-indoyl)ethyl phenyl keto ne in propylene glycol/buffer glasses, at 120 K. Both steady-state and kinetic data are satisfactorily fitted by an exponential distance dep endence of the rate, k(r) =; k(0) exp[-(r - r(0))/r(e)], with a contac t rate k(0) = 1.2 x 10(8) s(-1) and an attenuation length r(e) = 0.29 Angstrom. For a phosphorescence lifetime of 5 s, this rate yields an a verage interaction distance of 10 Angstrom. The rate is temperature de pendent, with k(0), estimated from the bimolecular quenching rate cons tant ((P)k(q)) of Trp analogues in liquids, increasing by about 10-fol d from 120 to 293 K. Solvent effects on the quenching efficiency are t ested with Trp analogues in water, propylene glycol, and dioxane. The quenching efficiency per collisional encounter is about 0.20 for water , 0.35 for propylene glycol, and drops to 0.025 in the aprotic, least polar dioxane. Acrylamide quenching rate constants are determined for a series of proteins and for experimental conditions appositely select ed to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to (P)k(q) = 1.5 x 10(9) M-1 s(-1) f or Trp in the solvent, the magnitude of (P)k(q) for protected Trp; res idues in proteins ranges from a maximum s of 6 x 10(4) M-1 s(-1), for the most superficial W59 of RNase T-1, to 10(-1) M-1 s(-1) for the mos t internal W109 of alkaline phosphatase. For most proteins, theoretica l estimates of (P)k(q) based on the distance dependence of the rate ex clude any quenching contribution from through-space: interactions by a crylamide in the solvent. This finding, together with a clear correlat ion between (P)k(q) and other indicators of molecular flexibility, imp lies that in the millisecond-second time scale of phosphorescence acry lamide can migrate through the macromolecule and that its rate is a me asure of the frequency and amplitude of the structural fluctuations un derlying diffusional jumps. The origin of the discrepancy between fluo rescence and phosphorescence quenching rates in proteins is discussed, and an alternative interpretation of fluorescence quenching data is p rovided.