DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

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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.