Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, koff, provide structural and functional 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 stable isotope 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. 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. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.
Overview of the MASE method
Theoretical modeling of subunit exchange at different koffvalues
Pan C (2011) Measuring Dissociation Rate Constants of Protein Complexes through Subunit Exchange: Experimental Design and Theoretical Modeling. PLoS ONE 6(12): e28827. doi:10.1371/journal.pone.0028827