Characterization of structural changes and large-scale protein dynamics and their influence on metal ion binding by human serum transferrin by ESI MS
Human serum Transferrin (hTf) is a ∼80 kDa protein, whose function is iron sequestration and transport. The two lobes of the protein (commonly referred to as N- and C-lobes) have a very high degree of structural identity and provide two distinct binding sites for a ferric ion. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells.
In the present study ESI MS is used to investigate interactions between human serum transferrin and two non-ferrous metals (indium and bismuth), conformational changes upon metal binding, as well as characterize human serum transferrin N-lobe (hTf/2N) global dynamics and functionally important local dynamic events. The In-hTf complex was directly detected by ESI MS; the Bi-hTf complex in solution was established by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination which causes anomalous instability of the transferrin-bismuth complex in the gas phase. The apo-hTf/2N and Fe-hTf/2N were used in hydrogen/deuterium exchange (HDX) measurement for characterizing protein dynamics. In this measurement, back-exchange was corrected for every transferrin N-lobe peptic fragment individually. The results showed that iron binding induce more compact conformation and significant decrease of HDX kinetics around hinge regions and Lys206 which is one amino acid from the dilysine-trigger. However, the changes around iron binding sites are not as significant. Our hypothesis is that instead of having frozen states, transferrin has certain frequency of conformational hopping.
A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures was developed utilizing the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode.