Monitoring lactoferrin iron levels by fluorescence resonance energy transfer: a combined chemical and computational study

Abstract

Abstract.Three forms of lactoferrin (Lf) that differed in their levels of iron loading (Lf, LfFe, and LfFe 2 ) 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 (LfFe 2 ). 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 LfFe 2 transition. This latter study demonstrated that lactoferrin supplies iron to this bacterium, and suggested that this process occurs with no protein internalization.

Evaluation of proteins in sheep colostrum via laser-induced breakdown spectroscopy and multivariate analysis

“…The very high affinity of lactoferrin for iron is relevant to its function against such microorganisms. In fact, lactoferrin can be considered as part of the immune system because of its effects on pathogens growth [30]. This antibacterial mechanism justifies clearly the results depicted in Fig.…”

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

“…Among several methods that are available in this context, Fluorescence Resonance Energy Transfer (FRET) technique has been widely used in vitro and in vivo to study protein-protein interactions (20,21,22,23,24). Since FRET efficiency is proportional to 1/r 6 , where "r" is the distance between the donor and the acceptor, the FRET signal provides a high degree of spatial sensitivity between 2 and 12 nm (7).…”

Chemically and Biologically Harmless versus Harmful Ferritin/Copper–Metallothionein Couples

“…Monoferric lactoferrin was purchased from Fonterra. Apolactoferrin (Lf) was prepared after following demetalation of the monomeric form as previously reported 27…”