Study On Conformation And Thermodynamics Of Calcium Ion - Induced EF - Hand Protein

As an important second messenger, calcium is involved in many cellular regulations; in which EF-hand proteins are functioning equally critical in these process. EF-hand proteins act as the Ca2+sensors, translating the chemical signal of an increased Ca2+concentration into diverse biochemical responses. Calcium and EF-hand proteins have been recognized as the key players in all aspects of cell function, starting with a cell’s birth during mitosis and ending with its apoptotic death. A malfunction in EF-hand protein signaling can engender several pathologies, such as cardiac hypertrophy, autoimmune disease, Alzheimer’s disease, Down’s syndrome and cancer.Calmodulin (CaM), the best studied example of an EF-hand calcium sensior protein, is involved in the regulation of many important Ca2+-dependent signaling pathways.calcineurin B (CNB), is the regulatory subunit of calcineurin, a Ca2+/CaM-dependent serine/threonine protein phosphatase that plays a critical role in many cellular processes. In this work, we focus on the conformational and thermodynamics changes of CaM and CNB induced by Ca2+binding, trying to reveal the interaction regularities between Ca2+and EF-hand proteins. The work includes the following three parts:1. The research background of EF-hand proteins, CNB and CaM are introduced detailedly. To study the interaction of Ca2+and EF-hand proteins by using Fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy and isothermal titration calorimetry (ITC) are suggested.2. The Ca2+-induced conformational changes in CaM were monitored by Fourier transform infrared spectroscopy (FT-IR) at different molar ratios of Ca2+to CaM. The results show that these changes occur in two distinctive transitions. The first transition involves significant changes in the overall secondary structure with a small gain in solvent accessibility, and is completed after the second Ca2+binds to both EF-hands of its C-terminal domain. The second transition is accompanied by CaM folding into a tighter, less hydrogen-exchangeable structure, and is completed by the addition of the fourth Ca2+to have four Ca2+per molecule. Particularly, a-helices in CaM-nCa2+(n=0,1,2) are less stable than those in CaM-nCa2+(n=3,4).3. The structural dynamics and thermodynamics of the interaction of Ca2+/Mg2+ and the calcineurin B domain (CNB) were monitored by Fourier transform infrared spectroscopy (FT-IR) and isothermal titration calorimetry (ITC). The results reveal that CNB activation by Ca2+binding involves significant conformational changes with a marked increase in the α-helix content, whereas Mg2+binds to CNB without inducing the changes in secondary structure that are characteristic of Ca2+binding. H-D exchange and GdnHCl-induced unfolding results show that the overall conformation of Ca2+-loaded CNB (CNB-Ca2+) is more stable and has more hydrophobic areas than its Ca2+-free CNB (apo-CNB) and Mg2+-loaded CNB (CNB-Mg2+). The thermodynamic characterization suggests that non-competition between Ca2+and Mg2+in their binding to the main CNB Ca2+binding sites. Mg2+more likely binds to the auxiliary cation-binding sites present on CNB.4. The research results of EF-hand proteins the recent research and discovery of the EF-hand protein have been reviewed and the prospect of related proteins have been put forward.