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

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 (τ1 = 1.7 ns, τ2 = 180 ps, and τ3 = 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 τ1 to Tyr-76 (R = 12.8 Å), τ2 to Tyr-115 (R = 6.9 Å), and τ3 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, Tm, obtained from Tyr-76 (Tm = 59.2°C) and Tyr-25 and Tyr-92 (Tm = 58.2°C) suggests a single unfolding event in this temperature range that affects all these regions similarly.