Istituto di Biofisica     
Cioni P., Strambini G. B. Acrylamide Quenching of Protein Phosphorescence as a Monitor of Structural Fluctuations in the Globular Fold. In: Journal of the American Chemical Society, vol. 120 (45) pp. 11749 - 11757. American Chemical Society, 1998.
This study examines acrylamide quenching of tryptophan room-temperature phosphorescence in proteins and the role that factors such as long-range 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 distance dependence of the through-space quenching rate is evaluated by studying the effects of acrylamide on the phosphorescence intensity and decay kinetics of the indole analogue 2-(3-indoyl)ethyl phenyl ketone in propylene glycol/buffer glasses, at 120 K. Both steady-state and kinetic data are satisfactorily fitted by an exponential distance dependence of the rate, k(r) = k0 exp[−(r − r0)/re], with a contact rate k0 = 1.2 108 s-1 and an attenuation length re = 0.29 . For a phosphorescence lifetime of 5 s, this rate yields an average interaction distance of 10 . The rate is temperature dependent, with k0, estimated from the bimolecular quenching rate constant (Pkq) of Trp analogues in liquids, increasing by about 10-fold from 120 to 293 K. Solvent effects on the quenching efficiency are tested 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 selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to Pkq = 1.5 109 M-1 s-1 for Trp in the solvent, the magnitude of Pkq for protected Trp residues in proteins ranges from a maximum of 6 104 M-1 s-1, for the most superficial W59 of RNase T1, to 10-1 M-1 s-1 for the most internal W109 of alkaline phosphatase. For most proteins, theoretical estimates of Pkq based on the distance dependence of the rate exclude any quenching contribution from through-space interactions by acrylamide in the solvent. This finding, together with a clear correlation between Pkq and other indicators of molecular flexibility, implies that in the millisecond-second time scale of phosphorescence acrylamide can migrate through the macromolecule and that its rate is a measure of the frequency and amplitude of the structural fluctuations underlying diffusional jumps. The origin of the discrepancy between fluorescence and phosphorescence quenching rates in proteins is discussed, and an alternative interpretation of fluorescence quenching data is provided.
DOI: 10.1021/ja9820543
Subject Phosphorescence

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