Study of the Dynamic Binding, Structure-Function of CIII

CIII的动态结合、结构-功能研究

• 批准号: 6677706
• 负责人: EMELITA D BREYER
• 金额: $ 13.22万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677706
• 关键词: SDS polyacrylamide gel electrophoresis; apolipoproteins; cardiovascular disorder risk; clinical research; enzyme linked immunosorbent assay; hepatic lipase; high density lipoproteins; human tissue; lipid metabolism; lipoprotein lipase; low density lipoprotein; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; scanning electron microscopy; site directed mutagenesis

DESCRIPTION (provided by applicant): Recent study shows that Apolipoprotein CIII (CIII) specifically binds and inhibits Lipoprotein Lipase (LpL) and Hepatic Lipase (HL) enzymes. The significance of the N-terminal domain in stabilizing the lipid-protein interaction and displacing Apolipoprotein E (E) was also identified (1). Our previous study that confirms the ability of CIII to displace E (2), sets up the stage for further structure-function studies evaluating the factors that enable CIII to displace E, and inhibits both HL and LpL. These functions should be investigated in correlation to CIIl's ability to bind to the lipoprotein particle (Lp) with varying size and composition. The cooperative and complex conformational modification of CIII binding to Lp and LpL/HL suggests that this study requires a factorial design experiment that can extract latent variable (factors) from collinear measurements of the protein's physico-chemical properties and structure (X-matrix), and relate these factors to the extracted latent variables from the Y-matrix consisting of the protein's functions. This study will be the first attempt to evaluate the structural factors in CIII that is responsible for the above functions in a systematic way using a factorial designed experiments. By using our model structure of CIII generated by homology with E crystal structure followed by molecular mechanics modeling, we are able to identify significant residues in CIII that may give us information regarding the difference in its function with respect to E and CII. Structural factors such as helix length, positive helix cap, loop flexibility, and hydrophobicity of side chains will be evaluated using different constructs in order to assess their contribution to the different functions of CIII. This approach is designed to give us a surface plot describing how the structural factors of CIII correlate with its functions. A more structured search and prediction of Clll analog that has a high lipid binding ability but with lipase inhibitory sites replaced by either the activating site of CII or E can be made using this approach.

描述（由申请人提供）： 最近的研究表明，载脂蛋白 CIII (CIII) 特异性结合并抑制脂蛋白脂肪酶 (LpL) 和肝脂肪酶 (HL)。 N 末端结构域在稳定脂质-蛋白质相互作用和取代载脂蛋白 E (E) 方面的重要性也得到了证实 (1)。我们之前的研究证实了 CIII 取代 E 的能力 (2)，为进一步的结构功能研究奠定了基础，以评估使 CIII 能够取代 E 的因素，并抑制 HL 和 LpL。应该研究这些功能与CIII1结合具有不同尺寸和组成的脂蛋白颗粒(Lp)的能力的相关性。 CIII 与 Lp 和 LpL/HL 结合的协同且复杂的构象修饰表明，这项研究需要因子设计实验，可以从蛋白质的物理化学性质和结构（X 矩阵）的共线测量中提取潜在变量（因子），并将这些因素与从由蛋白质功能组成的 Y 矩阵中提取的潜在变量相关联。本研究将是首次尝试使用析因设计实验来系统地评估 CIII 中负责上述功能的结构因素。通过使用与 E 晶体结构同源生成的 CIII 模型结构，然后进行分子力学建模，我们能够识别 CIII 中的重要残基，这可以为我们提供有关其功能相对于 E 和 CII 的差异的信息。将使用不同的构建体来评估结构因素，例如螺旋长度、正螺旋帽、环柔性和侧链疏水性，以评估它们对 CIII 不同功能的贡献。这种方法旨在为我们提供一个曲面图，描述 CIII 的结构因素如何与其功能相关。使用这种方法可以对具有高脂质结合能力但脂肪酶抑制位点被CII或E的激活位点取代的CII类似物进行更结构化的搜索和预测。