Characterization and Iron Binding Dynamics of Haemophilus Influenzae Ferric Binding Protein

Abstract

Iron is a fundamental metabolite for the survival of all biological systems because of its ability to function as both electron donor and acceptor. In unbound form, Fe+3 is not soluble in water whereas; Fe+2 is highly toxic in free form. Different organisms have developed specific mechanisms to transport iron. In humans transferrin is the responsible protein for carrying iron, whereas in bacteria Ferric Binding Protein (Fbp) performs this function. Pathogenic bacteria have evolved to sequester iron from human transferrin through an exceptional mechanism, where one of the two irons of transferrin is detached, transported through the periplasmic space and captured by Fbp. In this study, our main focus is to monitor conformational changes of Fbp from Haemophilus Influenzae, during iron release and binding in alternating environmental conditions. Fbp was expressed and purified by using recombinant DNA technology. Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), UV-Vis Absorption Spectroscopy (AS), Circular Dichroism (CD) and Small Angle X-ray Scattering (SAXS) are used for detecting structural changes. Additionally, features of the 480 nm ligand-to-metal charge transfer absorption peak of FBP-Iron complex was monitored during iron release/binding process for both Fe+3 and Fe+2 in different pH and ionic strength conditions. Our results confirm that Fbp changes its conformation to bind iron; hydrodynamic radius decreases when it captures the ligand because of the closing up of the metal-binding cleft. Effect of changes in pH, ionic strength and the state of iron on iron affinity are also investigated. We believe that the study on the dynamics of Fbp addresses us to further biosensor applications to determine elevated levels of iron in individual cells and targeted drug design practices.