Influence of denaturants on native-state structural fluctuations in azurin probed by molecular size-dependent quenching of Trp phosphorescence.

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.

Amplitude spectrum of structural fluctuations in proteins from the internal diffusion of solutes of increasing molecular size: a Trp phosphorescence quenching study.

The accessibility of O(2), acrylamide, and four acrylamide derivatives of increasing molecular size {N-(hydroxymethyl)acrylamide, N,N'-methylene-bisacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid} to buried Trp residues in four proteins, as determined by dynamic quenching of their phosphorescence emission, was utilized for probing the amplitude range of structural fluctuations in these macromolecules. The quenching rate constant of each solute, k(q), was determined (at 25 and -5 °C) for liver alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, azurin, and alkaline phosphatase. The results show that high-frequency small amplitude motions pervade the protein globular fold, permitting relatively unhindered diffusion of small diatomic molecules all the way to compact cores of the macromolecule.

Acrylonitrile quenching of trp phosphorescence in proteins: a probe of the internal flexibility of the globular fold.

Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O(2) and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone.

Protein phosphorescence quenching: distinction between quencher penetration and external quenching mechanisms.

The accessibility of quenching solutes, Q, of various molecular sizes to buried Trp residues in proteins, as attested by dynamic quenching of their phosphorescence emission, is instrumental for probing structural fluctuations in these macromolecules. However, interpretation of quenching rates in terms of Q migration through the globular fold requires that alternative reaction pathways, such as long-range interactions with Q in the solvent, be ruled out. In theory, the external quenching rate can be estimated from the distance dependence of the through-space interaction by assuming compliance with the rapid diffusion limit regime.

Fluorescence quenching of buried Trp residues by acrylamide does not require penetration of the protein fold.

The accessibility of acrylamide to buried Trp residues in proteins, as attested by dynamic quenching of their fluorescence emission, is often interpreted in terms of migration of the quencher (Q) through the globular fold. The quencher penetration mechanism, however, has long been debated because, on one hand, solutes the size of acrylamide are not expected to diffuse within the protein matrix on the nanosecond time scale of fluorescence and, on the other hand, alternative reactions pathways where Q remains in the solvent cannot be ruled out. To test the Q penetration hypothesis, we compared the quenching rates of acrylamide analogs of increasing molecular size (acrylonitrile, acrylamide, and bis-acrylamide) on the buried Trp residues of RNaseT1 and parvalbumin.