SELF ASSOCIATION OF PROTEINS (SIMSTEX): DEVELOPMENT & APPLICATON

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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. Protein-protein interactions are crucial for many biological functions in living systems Disruptions in these interactions can lead to various diseases including metabolic disorders, cancer and infections. We are interested in developing mass spectrometric methods that take advantage of the high sensitivity and specificity of mass spectrometry, combined with H/D exchange and mathematical modeling to study self association. Insulin, a 51-amino acid protein is used in the treatment of diabetes. The tendency of human insulin to self associate to form hexamers in solution when reduced by mutating key residues gives faster-acting insulin analogs. Self association Interactions using Mass Spectrometry, self Titration and amide EXchange (SIMSTEX), a technique we developed to obtain affinity constants for oligomerization from titration data was applied to self association of insulin and insulin analogs. The affinity constants, we calculated, agree within a factor of 20 to the literature values for human and lispro insulins. The trends we observed for insulin mutants, also, agree well with literature reports, establishing SIMSTEX as a reliable technique to study self association.