title: Measuring dissociation rate constants of protein complexes through subunit exchange: experimental design and theoretical modeling.
abstract: Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off), provide structural and functional information on protein complexes. However, because all existing methods for measuring k(off) 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 k(off) 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 k(off) 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.
authors.affiliation: Oak Ridge National Laboratory, Computer Science and Mathematics Division and BioSciences Division, Oak Ridge, Tennessee, United States of America. panc@ornl.gov
authors.forename: Chongle
authors.initials: C
authors.lastname: Pan
mesh_terms: D007700:Kinetics; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D012107:Research Design; D013194:Staining and Labeling
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits