DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

蛋白质生物物理学 H/D 交换的发展

• 批准号: 7355150
• 负责人: MICHAEL L GROSS
• 金额: $ 1.45万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355150
• 关键词: DEVELOPMENT; EXCHANGE; PROTEIN; BIOPHYSICS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Quantification of Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange (PLIMSTEX) is a newly developed method from this Research Resource for determining the binding stoichiometry and affinity of a wide range of protein-ligand interactions. In this research, we are developing a method for analyzing the PLIMSTEX titration curves and evaluate the effect of various models on the precision and accuracy for determining binding constants using H/D exchange and a titration. The titration data can be fitted using a 1:n protein:ligand sequential binding model, where n is the number of binding sites for the same ligand. An ordinary differential equation was used for the first time in calculating the free ligand concentration from the total ligand concentration. A non-linear least squares regression method was applied to minimize the error between the calculated and the experimentally measured deuterium shift by varying the underlying parameters. A sub-sampling method and second-order statistics we re used to evaluate the uncertainties of the fitting parameters. The interaction of intestinal fatty-acid-binding protein (IFABP) with a fatty-acid carboxylate and that of calmodulin with Ca2+ have been used as two tests. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses novel aspects in modeling experimental titration data to determine the affinity of ligand binding. We expect to continue to develop and evaluate the model in future core research.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。通过质谱，滴定和H/D交换（PLIMSTEX）定量蛋白质配体相互作用是一种新开发的方法，用于确定广泛的蛋白质-配体相互作用的结合化学计量和亲和力。在这项研究中，我们正在开发一种分析PLIMSTEX滴定曲线的方法，并评估各种模型对使用H/D交换和滴定法测定结合常数的精密度和准确度的影响。滴定数据可以使用1：n蛋白质：配体顺序结合模型拟合，其中n是相同配体的结合位点数。一个常微分方程首次用于计算游离配体浓度从总配体浓度。采用非线性最小二乘回归方法，通过改变基本参数，使计算值和实验测量值之间的误差最小化。采用子抽样方法和二阶统计量对拟合参数的不确定度进行了估计。肠脂肪酸结合蛋白（IFABP）与脂肪酸羧酸盐的相互作用和钙调素与Ca 2+的相互作用已被用作两个测试。这里描述的建模过程不仅是一个新的工具，用于分析的H/D交换数据ESI-MS获得，但也具有新的方面，在模拟实验滴定数据，以确定配体结合的亲和力。我们希望在未来的核心研究中继续开发和评估该模型。