SELF ASSOCIATION OF PROTEINS (SIMSTEX): DEVELOPMENT & APPLICATON

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。蛋白质-蛋白质相互作用对生命系统中的许多生物功能至关重要，这些相互作用的破坏可导致各种疾病，包括代谢紊乱、癌症和感染。我们有兴趣开发质谱仪方法，利用质谱的高灵敏度和特异性，结合H/D交换和数学建模来研究自关联。胰岛素是一种含有51个氨基酸的蛋白质，用于治疗糖尿病。人胰岛素在溶液中自我结合形成六聚体的趋势，当通过突变关键残基产生快速作用的胰岛素类似物时。利用质谱法、自滴定法和酰胺交换（SIMSTEX）的自关联相互作用，我们开发了一种从滴定数据中获得寡聚化亲和常数的技术，应用于胰岛素和胰岛素类似物的自关联。我们计算出的亲和常数与人类胰岛素和lispro胰岛素的文献值相差不到20倍。我们在胰岛素突变体中观察到的趋势也与文献报道一致，建立了SIMSTEX作为研究自我关联的可靠技术。

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.