title: Influence of denaturants on native-state structural fluctuations in azurin probed by molecular size-dependent quenching of Trp phosphorescence.
abstract: Using azurin as a model protein, this study enquires on the nature of small and large amplitude structural fluctuations permitting the penetration of different size solutes within protein folds, as inferred from quenching of the phosphorescence of buried Trp residues. The work examines the effect that guanidinium hydrochloride and urea have on the migration of a range of quencher molecules of increasing molecular size (M(w) range = 32-206 Da). Using the quenching rate constant of Trp phosphorescence as a monitor, the results demonstrate that structural fluctuations linked to O(2) migration are not affected by denaturants, whereas larger amplitude structural fluctuations necessary to facilitate penetration of bulkier quencher molecules [acrylonitrile, acrylamide, N-(hydroxymethyl) acrylamide, N-[tris(hydroxymethyl) methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid] are clearly enhanced by the presence of denaturant. These data when interpreted in the context of the amount of new protein surface uncovered to solvent (ΔSASA(o)) by the underlying structural fluctuations show a direct correlation between the amplitude of these motions and ΔSASA(o). Denaturants also promote slow frequency (20-80 s(-1)) conformational transitions, not manifested in normal aqueous solutions, which provide extra migration pathways for acrylamide and its larger derivatives. The quencher-size dependence of the quenching rate provides evidence of multiple, independent quencher migration pathways to the azurin core, which are characterized by motions on different time scales, microseconds and milliseconds, and by a 3- to 5-fold difference in ΔSASA(o), respectively.
authors: Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy. strambini@pi.ibf.cnr.it
authors: Giovanni B
authors: GB
authors: Strambini
authors: Margherita
authors: M
authors: Gonnelli
mesh_terms: D001400:Azurin / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
chemical_list: D001400:Azurin; D014364:Tryptophan; D014508:Urea; D019791:Guanidine