Intramolecular Fluorescence Quenching of Tyrosine by the Peptide α-Carbonyl Group Revisited

Abstract

The environment-dependent multiexponential behavior of N-acetyltyrosinamide (NAYA) and other tyrosine derivatives is revisited, aiming for a better understanding of tyrosine as an intrinsic fluorescent probe for protein microenvironment changes during conformational changes. The effects of solvent polarity, viscosity, and temperature on the fluorescence decay of NAYA were evaluated using dioxane-water mixtures and pure solvents. Double-exponential decays were observed in dioxane-water mixtures above 70% (v/v) water concentration including pure water, for temperatures below 50 °C. However, at higher temperatures, or in dioxane-water mixtures with lower water concentrations, NAYA shows single-exponential decays. Singleexponential decays were also generally observed in pure solvents (dioxane, acetonitrile, methanol, ethanol, DMSO). The exception was the strong hydrogen-bond donor trifluorethanol, in which NAYA decays as a double exponential. The results are consistent with a solvent-modulated excited-state intramolecular electron transfer from the phenol to the amide moiety occurring in one of the three rotamers of NAYA. On the basis of a full kinetic analysis of the data, it is shown that experimental observation of double-exponential decays depends on three factors: the ground-state population of rotamers, their rates of interconversion (k r ) 4.4 × 10 8 s -1 and k r ′ ) 5.2 × 10 8 s -1 , in water at 23 °C, E r ) 4.9 ( 0.1 kcal mol -1 and E r ′) 5.2 ( 0.3 kcal mol -1 ), and the electron-transfer rate constant (k ET ) 2.0 × 10 9 s -1 , in water at 23 °C, E ET ) 1.8 ( 0.2 kcal mol -1 ). Solvent viscosity controls the interconversion rate constants while solvent polarity and hydrogen-bonding ability determines the Gibbs energy of electron transfer and the magnitude of its rate constant. Consequently, the nature of tyrosine decays in proteins is determined from a delicate balance between the interconversion and electron-transfer rate constants.

Conformational Heterogeneity of a Leucine Enkephalin Analogue in Aqueous Solution and Sodium Dodecyl Sulfate Micelles: Comparison of Time-Resolved FRET and Molecular Dynamics Simulations

“…The theory of tyrosyl rotamer interchange and its effect on the fluorescence decay of the tyrosyl residue in small peptides has been described in detail by us and others 23,31,32. That work demonstrates definitively that rotamer interchange occurs on the time scale of the fluorescence decay and must be accounted for to accurately describe the photophysics of Tyr in peptides.…”

“…In order to calculate the FRET efficiency, it was therefore necessary to know the fluorescence lifetime of the energy donor (Tyr) in the absence of FRET. But the Tyr lifetime depends strongly on the Tyr rotameric configuration 23,31,32. Therefore, it was necessary to take into account the rotameric configuration of Tyr (but not of Trp, because it is the energy acceptor and its lifetime was not measured) to determine the FRET rate.…”

Solvent Effects on the Fluorescence Quenching of Tryptophan by Amides via Electron Transfer. Experimental and Computational Studies

“…26,27 Noronha et al 27 reported that N-acetyltyrosinamide has Φ f ) 0.18 in dioxane, and drops to 0.05 as the water composition of the solvent is increased to 100% at 25°C.…”

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles

“…In order to compare the measured and calculated transfer efficiencies, the individual tyrosine quantum yields must be known. It is possible that some of the tyrosine’s are quenched by interactions with near-by polar groups [35]. For example, in the belayed structure of AB, Tyr 43 is hydrogen-bonded to Lees 30, so it is unlikely to emit.…”