Kinetics of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing and Fluorescence Stoichiometry.

Low-affinity protein complexes and their transient states are difficult to measure in single-molecule experiments because of their low population at low concentrations. A prominent solution to this problem is the use of microfluidic mixing devices, which rely on diffusion-based mixing. This is not ideal for multiprotein complexes, as the single-molecule fluorescence signal is dominated by the already dissociated species. Here, we designed a microfluidic device with mixing structures for fast and homogeneous mixing of components with varying diffusion coefficients and for fluorescence measurements at a defined single-molecule concentration. This enables direct measurement of dissociation rates at a broad range of timescales from a few milliseconds to several minutes. This further allows us to measure structural properties and stoichiometries of protein complexes with large equilibrium dissociation constants ( KD's) of 5 μM and above. We used the platform to measure structural properties and dissociation rates of heat shock protein 90 (Hsp90) dimers and found at least two dissociation rates which depend on the nucleotide state. Finally, we demonstrate the capability for measuring also equilibrium dissociation constants, resulting in the determination of both the kinetics and thermodynamics of the system under investigation.