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

Melinda Noronha ^* , João C. Lima , Emanuele Paci , Helena Santos  and António L. Maçanita ^*

^* Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisbon, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; REQUIMTE/CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal; Department of Physics and Astronomy, University of Leeds, Leeds, United Kingdom

Correspondence: Address reprint requests to António L. Maçanita, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Tel.: 351-21-8419606; E-mail: macanita{at}ist.utl.pt.

The six tyrosine residues of ribonuclease A (RNase A) are used as individual intrinsic probes for tracking local conformational changes during unfolding. The fluorescence decays of RNase A are well described by sums of three exponentials with decay times (₁ = 1.7 ns, ₂ = 180 ps, and ₃ = 30 ps) and preexponential coefficients (A₁ = 1, A₂ = 1, and A₃ = 4) at pH 7, 25°C. The decay times are controlled by photo-induced electron transfer from individual tyrosine residues to the nearest disulphide (–SS–), bridge, which is distance (R) dependent. We assign ₁ to Tyr-76 (R = 12.8 Å), ₂ to Tyr-115 (R = 6.9 Å), and ₃ to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 ± 0.3 Å) at 23°C. On the basis of this assignment, the results show that, upon thermal or chemical unfolding only Tyr-25, Tyr-92, and Tyr-76 undergo significant displacement from their nearest –SS– bridge. Despite reporting on different regions of the protein, the concordance between the transition temperatures, T_m, obtained from Tyr-76 (T_m = 59.2°C) and Tyr-25 and Tyr-92 (T_m = 58.2°C) suggests a single unfolding event in this temperature range that affects all these regions similarly.