Abstract
Protein complexes are generally dynamic macromolecules that constantly disintegrate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, koff, provide important structure and function information on protein complexes. However, because all existing methods for measuring koff require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE) to measure koff using metabolic labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. In this study, the subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that koff determines, and hence can be estimated from, the observed rate of subunit exchange. Future study will be needed to experimentally validate the new method.