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

¹ Instituto Superior Técnico
² Universidade Nova de Lisboa
³ University of Leeds
⁴ Instituto de Tecnologia Química e Biológica

^* To whom correspondence should be addressed. E-mail: macanita{at}ist.utl.pt.

Submitted on July 19, 2006
Revised on October 9, 2006
Accepted on 1 February 2007

	Abstract

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 3 exponentials with decay times (1 = 1.7 ns, 2 = 180 ps and 3 = 30 ps) and pre-exponential 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.

Key Words: Disulphide bridges, Electron transfer, Fluorescence decays, MD simulations, Protein unfolding