DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。用质谱仪、滴定和H/D交换法定量测定蛋白质配体相互作用(PLIMSTEX)是本研究资源中发展起来的一种新方法，用于确定广泛的蛋白质-配体相互作用的结合化学计量比和亲和力。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H/D交换和a滴定测定结合常数的精密度和准确度的影响。滴定数据可以用1：N蛋白质：配体顺序结合模型来拟合，其中n是同一配体的结合位点数。首次使用常微分方程式从总配体浓度计算自由配体浓度。采用非线性最小二乘回归方法，通过改变基础参数来最小化计算结果与实验测量结果之间的误差。采用二阶统计量和二次抽样方法对拟合参数的不确定度进行了评估。肠脂肪酸结合蛋白(IFABP)与脂肪酸羧酸盐的相互作用和钙调蛋白与钙离子的相互作用被用作两个实验。本文描述的建模过程不仅为分析ESI-MS获得的H/D交换数据提供了一种新的工具，而且在模拟实验滴定数据以确定配体结合亲和力方面也具有新的方面。我们希望在未来的核心研究中继续开发和评估该模型。

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.