Unfolding of Ubiquitin Studied by Picosecond Time-Resolved Fluorescence of the Tyrosine Residue

Abstract

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25 degrees C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6 degrees C, DeltaHTm = 56.5 kcal/mol, and DeltaCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0 degrees C, DeltaHm= 51.4 kcal/mol, and DeltaCp = 730 cal/mol//K.

Tracking Local Conformational Changes of Ribonuclease A Using Picosecond Time-Resolved Fluorescence of the Six Tyrosine Residues

“…Above this water content, a second shorter decay time (;400 ps) starts to develop and its normalized preexponential ultimately reaches the value of 0.12 in water (9). Very similar observations were made with the single-tyrosine protein UBQ, but the preexponential value was greater (5). The observation of this double-exponential decay, which results from electron transfer from one of the three cresol rotamers to the protein backbone, depends on three factors: i), the relative mol fractions of the ground-state rotamer population, the rotamer conformations being determined by rotation about the C a -C b bond (a); ii), the rotational rate constants for interconversion between the rotamers, k r and k r 9; and iii), the photo-induced electron transfer rate constant, k ET , which depends strongly on solvent polarity (9).…”

“…Studies carried out by Wills and Szabo (19) demonstrated that the fluorescence of Tyr is quenched in the presence of hydrogen-bond acceptors such as acetate. We have made a similar observation for Tyr in UBQ, whose fluorescence is quenched as a result of proton transfer to a nearby carboxylate group of Glu-51 (5). Since previous studies have suggested that, at pH 5, Tyr-25 of RNase A is hydrogen-bonded to the carboxylate of Asp-14 (7,20), we have analyzed the fluorescence response to the pH change.…”

“…Among the fluorescence quenching mechanisms known for tyrosine within proteins, quenching by proton transfer to a carboxylate of an aspartic or glutamic amino acid residue has been observed in bovine ubiquitin (5). Studies have suggested that Tyr-25 is hydrogen bonded to Asp-14.…”

“…The use of TRFS has been hindered, in part, because several studies have shown the appearance of more than one decay time for proteins with a single Trp (2) or Tyr (3) residue. However, we have recently shown that the fluorescence of Staphylococcus aureus nuclease A (SNase), with a single Trp (4), and that of bovine ubiquitin (UBQ), with a single tyrosine (Tyr) residue (5), decay as a single exponential below the unfolding temperature region. Above this temperature region, the decays become multiexponential due to the fluorescence contribution of the unfolded protein, and from the preexponential coefficients of these decays, the mol fractions of the folded and unfolded proteins could be accurately evaluated and used to calculate equilibrium constants, enthalpies, entropies, and specific heats of unfolding.…”

Regulation of centrin self-assembly investigated by fluorescence resonance light scattering

“…The proton of Tyr in the excited state may be transferred to carboxyl of the Glu residue or Asp residue, if the two residues are located near Tyr. 34 At pH 7.4, Ser166 of EoCen can be phosphorylated by PKA. The introduced phosphate group at Ser166 located near Tyr168 becomes negatively charged.…”