Calmodulin의 IP3 수용체 Suppressor 도메인 및 Lysozyme과의 결합에 있어서의 기능적 기전적 연구

Title
Calmodulin의 IP3 수용체 Suppressor 도메인 및 Lysozyme과의 결합에 있어서의 기능적 기전적 연구
Authors
강선미
Keywords
calmodulin의ip3수용체suppressor도메인및lysozyme과의결합에있어서의기능적기전적연구
Issue Date
2012
Publisher
인하대학교
Abstract
Ca2+ plays essential roles in various types of cell signaling. One of the important Ca2+ channels is the inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R regulates the calcium flux in and out of the endoplasmic reticulum (ER), where intracellular Ca2+ is stored. The IP3R opens when IP3 binds to the ligand binding domain of the IP3R. Various proteins and molecules such as IRBIT, ERp44, and CaBP also interact with the IP3R, which causes the channel to close. Among them CaM and Ca2+ are key regulators of the IP3R in helping the IP3R maintain cytosolic Ca2+ concentration. Although the roles of CaM and Ca2+ in the regulation of Ca2+ release have been reported, the precise mechanisms and structural information remain unknown. To address these issues, we studied the interactions between CaM and the suppressor domain of IP3R using pull down, NMR, circular dichroism (CD), and fluorescence experiments. The results indicate that apo-CaM induces large-scale conformational changes in the supp
Description
General Introduction 1 Part 1. Functional and Structural studies on the Interaction between IP3 receptor Suppressor domain and Calmodulin 5 1. Introduction 5 1-1. Calcium 5 1-2. IP3R 5 1-3. CaM binding sites within IP3R 14 2. Materials and methods 15 2-1. Materials 15 2-2. Cloning of CaM and IP3R suppressor domain gene 15 2-3. Expression and purification of CaM (CaM mutant) and IP3R suppressor domain 18 2-4. Pull down 19 2-5. NMR 19 2-6. Fluorescence spectroscopy 20 2-7. Circular dichroism (CD) spectroscopy 20 3. Results 21 3-1. Protein expression and purification of the His-CaM 21 3-2. Protein expression and purification of the IP3R suppressor domain 21 3-3. Binding experiment using affinity column 21 3-4. Accessibility of CaM binding sites within suppressor domain 22 3-5. NMR spectroscopy of labeled IP3R suppressor domain 29 3-6. NMR spectroscopy of labeled CaM 35 3-7. Prediction of secondary structure changes using circular dichroism (CD) spectroscopy 36 3-8. Calculation of Kd with dansyl-CaM fluorescence 43 4. Discussion 47 Part 2. Functional and Structural studies on the Interaction between Calmodulin and Lysozyme 54 1. Introduction 54 1-1. Lysozyme 54 1-2. CaM and lysozyme 55 2. Materials and methods 56 2-1. Materials 56 2-2. Expression and purification of CaM 56 2-3. Pull down assay 57 2-4. Native gel electrophoresis 57 2-5. NMR 57 3. Results 59 3-1. Characterization of the binding between CaM and lysozyme 59 3-2. Identification of binding sites in CaM using NMR 62 4. Discussion 65 5. References 72
URI
http://dspace.inha.ac.kr/handle/10505/23457
Appears in Collections:
Medical School/College of Medicine (의학전문대학원/의과대학) > Medical Science (의학) > Theses(의학 석박사 학위논문)
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