Fluorescence Lifetimes of Tyrosine Residues in Cytochrome c as Local Probes to Study Protein Unfolding

Time-resolved fluorescence spectroscopy was used to show that multiple tyrosine residues of a protein can serve as localized probes of structural changes during thermal unfolding. Cytochrome c from Methylophilus methylotrophus, which has four tyrosine residues, was chosen as a model protein. The procedure involved, first, the assignment of the experimental decay times to the tyrosine residues, followed by the interpretation of the changes in the decay times and pre-exponential coefficients with temperature. We found that the fluorescence decays of cytochrome c are double-exponential from 23 to 80 degrees C, with decay times much shorter than those of the parent compound N-acetyl-tyrosinamide; this quenching was ascribed to dipole-dipole energy transfer from the tyrosine residues to the heme. The tyrosine-heme distances (R) and theoretical decay times, tau(comp), were estimated for each tyrosine residue. The analysis of the simulated decay generated with tau(comp), showed that a double-exponential fit is sufficient to describe the four decay times with two pre-exponential coefficients close to values observed from the experimental decay. Therefore, the decay times at 23 degrees C could be assigned to the individual tyrosine residues as tau(1) to Tyr-10 and Tyr-23 (at 20.3 angstrom) and tau(2) to Tyr-12 and Tyr-115 (at 12-14 angstrom). On the basis of this assignment and MD simulations, the temperature dependence of the decay times and pre-exponential coefficients suggest that upon unfolding, Tyr-12 is displaced from the heme, with loss of the structure of alpha-helix I. Moreover., Tyr-115 remains close to the heme and the structure in this region of the protein is not altered significantly. Altogether the data support the view that the protein core, comprising the heme and the four alpha-helices II to V, is clearly more stable than the remaining region that includes alpha-helix I and the loop between residues 19-27.