The Kinetics Of Calmodulin Induced By Calcium Ion And Thermal Dynamics Research

As an important second messenger, calcium ion (Ca2+) participates in a number of physiological responses both in animal and plant cells. As a simple ion signaling molecule, the downstream receptor (calcium receptor protein) is very important if the Ca2+wants to pass the signal along and adjust a variety of biochemical and cellular responses. The Ca2+-binding protein calmodulin (CaM) is the single most important transducer of intracellular Ca2+signals. It is involved in all aspects of cardiovascular function through its many target proteins, which include adenylate cyclades, numerous protein kinases, the protein phosphatase calcineurin (CN), nitric oxide synthase, and several channels, notably GAP (GTPase activating protein) junctions, voltage-gated Ca2+channels, and SK potassium channels. CaM can bind to and regulate a variety of different target proteins, thereby affecting many different cellular functions, such as inflammation, metabolism, apoptosis, muscle contraction, intracellular movement, short-term and long-term memory, nerve growth and immune response. Many CaM-binding proteins are unable to combine calcium themselves, so CaM as a calcium sensor conducts signal transduction.Calcium-binding protein CaM belongs to the EF-hand family with four EF-hand domains, which can bind four calcium ions. CaM undergoes a conformational change upon binding to calcium, which enables bind to specific proteins for a specific response. The alteration of the Ca2+-binding affinities of these proteins strongly influence their functions in Ca2+homeostasis, including uptake, transport, and maintenance of the proper Ca2+concentration in the cellular environment. One of the most intriguing questions about Ca+signaling is how EF-hand proteins use the ubiquitous EF-hand Ca2+-binding motif to coordinate diverse cellular functions with different Ca2+-binding affinities; especially how Ca2+binding regulates the biological functions of trigger/sensor proteins, such as CaM, in the Ca2+-signaling cascade.In this work, a series of work has been done especially on the CaM conformational changes in the role of Ca2+, which includes the following three parts: First, the research background of structure and function of CaM was introduced and illustrated. Second, GdnHCl-induced unfolding of CaM by monitoring changes in ellipticity at222nm using Far-UV CD at various concentrations of GdnHCl were examined. The data revealed that the GdnHCl-induced unfolding of Holo-CaM is less cooperative than that of Apo-CaM. We explored the conformational and structural dynamics of CaM using amide hydrogen deuterium (H-D) exchange coupled by Fourier transform infrared (FT-IR) spectroscopy. The results of H-D exchange and FT-IR suggest that CaM activation by Ca2+binding involves significant conformational changes. Quantitative analysis by curve-fitting revealed upon Ca2+binding, significant changes in the amide I region were observed, particularly a marked increase in the a-helix band intensity, which coincides with disappearance of the band at1646cm-1(random coil). This observation is consistent with the molecular model established on the basis of X-ray crystallography data. The thermodynamics of the interaction between Ca2+and CaM was examined using isothermal titration calorimetry (ITC). The molar ratio of Ca2+to CaM is consistent with the results reported for nuclear magnetic resonance spectroscopy, at the same time, we accessed to the Ca2+and CaM binding constant Ka. The EF-hand motifs of CaM contain12amino acid residues in the Ca2+-binding loop and have been proposed to undergo a two-step Ca2+-binding process. The ITC method provides a new strategy for obtaining site-specific Ca2+binding properties and a better estimation of the cooperatively and conformational change contributions of coupled EF-hand proteins. Third, each CaM molecules combines with four calcium ions, however, the role of each Ca2+ion in the binding process is different. We studied the CaM conformational change process with hydrogen-deuterium exchange method under conditions of different concentrations of calcium. By H-D exchange experiments, significant changes were observed in the overall secondary structure. The secondary structure of CaM was clearly reflected, which indicated that after the second calcium binding the CaM began to stabilize. Fourth, we conducted a preliminary study of CaM target protein of CN.