Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue

• Type: Article in International Scientific Journal
• Date: 2004

Authors

Noronha, M (Author) Other; Lima, JC (Author) Other; Bastos, M (Author) FCUP; Santos, H (Author) Other; Macanita, AL (Author) Other

Journal

Title: Biophysical Journal

Vol. 87

Pages: 2609-2620

ISSN: 0006-3495

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 20 Citations

Scopus - 21 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-866

DOI: 10.1529/biophysj.104.046466

Abstract (EN): 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 (25degreesC) 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 (K-U) 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 T-m=54.6degreesC, DeltaH(Tm)=56.5 kcal/mol, and DeltaC(p)=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, T-m=57.0degreesC, DeltaH(m)=51.4 kcal/mol, and DeltaC(p)=730 cal/mol//K.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12