Excited-state lifetime studies of the three tryptophan residues in the N-lobe of human serum transferrin

Abstract

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N-lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N-lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues. Decays of these samples are best described by two or three discrete lifetimes or by a unimodal Lorentzian distribution. The discrete lifetimes and the center of the lifetime distribution for samples containing Trp128 and Trp264 are affected by iron. The distribution width narrows on iron removal and is consistent with a decrease in dynamic mobility of the dominant fluorophore, Trp264. Both the quantum yield and the lifetimes are lower when iron is present, however, not proportionally. The greater effect of iron on quantum yields is indicative of nonexcited state quenching, i.e., static quenching. The results of these experiments provide quantitative data strongly suggesting that Förster resonance energy transfer is not the sole source of Trp quenching in the N-lobe of hTF.

Figure 1

Crystal structures of hTF N-lobe in both the iron bound (A [PDB ID: 1A8E]) and apo (B [PDB ID: 1BP5]) states showing the location of each of the three Trp residues (highlighted in red) and the large conformational change in the N-lobe associated with iron removal. This figure was made using PyMol. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

Steady-state emission spectra of iron bound (A) and apo (B) WT and the three single point Trp mutants. Overlay of the WT N-lobe and the three double mutants in iron bound (C) and apo (D) states. Emission scans were generated using 300 nm as the excitation wavelength and monitoring between 310 and 450 nm.

Figure 3

Lorentzian distribution of apo and iron bound lifetimes overlaid with the discrete components: (A) WT N-lobe, (B) W8Y, (C) W128Y, (D) W264Y, (E) W8, (F) W128, and (G) W264. Each Lorentzian distribution was calculated with one discrete component.

Figure 4

Positions of hTF N-lobe Trp from apo (PDB ID: 1BP5) and iron bound (1A8E) structures. (A) Close-up view of Trp8 surrounded by two disulfide bonds (Cys9/Cys48 and Cys19/Cys39), Val60, Thr61, and Phe295 and two water molecules (red spheres). (B) Close-up view of the change in local environment of Trp128 in the iron bound and apo states. (C) Close-up view of the local environment of Trp264. This figure was made using PyMol. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]