Monitoring lactoferrin iron levels by fluorescence resonance energy transfer: a combined chemical and computational study.
Three forms of lactoferrin (Lf) that differed in their levels of iron loading (Lf, LfFe, and LfFe2) were simultaneously labeled with the fluorophores AF350 and AF430. All three resulting fluorescent lactoferrins exhibited fluorescence resonance energy transfer (FRET), but they all presented different FRET patterns. Whereas only partial FRET was observed for Lf and LfFe, practically complete FRET was seen for the holo form (LfFe2). For each form of metal-loaded lactoferrin, the AF350-AF430 distance varied depending on the protein conformation, which in turn depended on the level of iron loading. Thus, the FRET patterns of these lactoferrins were found to correlate with their iron loading levels. In order to gain greater insight into the number of fluorophores and the different FRET patterns observed (i.e., their iron levels), a computational analysis was performed. The results highlighted a number of lysines that have the greatest influence on the FRET profile. Moreover, despite the lack of an X-ray structure for any LfFe species, our study also showed that this species presents modified subdomain organization of the N-lobe, which narrows its iron-binding site. Complete domain rearrangement occurs during the LfFe to LfFe2 transition. Finally, as an example of the possible applications of the results of this study, we made use of the FRET fingerprints of these fluorescent lactoferrins to monitor the interaction of lactoferrin with a healthy bacterium, namely Bifidobacterium breve. This latter study demonstrated that lactoferrin supplies iron to this bacterium, and suggested that this process occurs with no protein internalization.
Departamento de Química Inorgánica, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, 18071, Granada, Spain.
Fernando
F
Carmona
Víctor
V
Muñoz-Robles
Rafael
R
Cuesta
Natividad
N
Gálvez
Mercè
M
Capdevila
Jean-Didier
JD
Maréchal
José M
JM
Dominguez-Vera
D019295:Computational Biology / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007501:Iron / Q000032:analysis* / Q000737:chemistry*; D007781:Lactoferrin / Q000032:analysis* / Q000737:chemistry*; D017433:Protein Structure, Secondary; D014961:X-Ray Diffraction
C490098:LTF protein, human; D007501:Iron; D007781:Lactoferrin