Many protein complexes are assembled from a varying number of subunits, which are continuously exchanging with diverse time scales. This structural dynamics is considered to be important for many regulatory and sensory adaptation processes that occur . We have developed an accurate method for monitoring protein subunit exchange by using native electrospray ionization mass spectrometry (ESI-MS), exemplified here for an extremely stable Rad50 zinc hook (Hk) dimer assembly, Zn(Hk). The method has two steps: appropriate protein/peptide mutation and native ESI-MS analysis using a variable-temperature sample inlet. In this work, two Hk mutants were produced, mixed with wild-type Hk, and measured at three different temperatures. A thermokinetic analysis of heterodimer formation allowed us to determine the enthalpy, entropy, and Gibbs free energy of activation for subunit exchange, showing that the reaction is slow and associated with a high enthalpic barrier, consistent with the exceptionally high stability of the Zn(Hk) assembly.
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