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. The propensity of various insulins and their analogs to oligomerize can be investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of assocn. in soln. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titrn., and H/D exchange) to det. protein/ligand affinities in soln., to det. self-assocn. equil. consts. for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-assocn. interactions using mass spectrometry, self-titrn. and H/D exchange); it gives affinity consts. that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-assoc., possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-assocn., thus providing potentially faster-acting analogs for use in controlling diabetes.