Single-Molecule Fluorescence Measurements of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing

Abstract

The dynamic assembly and disassembly of multi-protein complexes is an integral part of many signaling processes. While thermodynamic parameters like the equilibrium rate constants, KD's, are relatively easily determined, the full kinetics is much harder to access. This is particularly the case for fast kinetics of protein complexes consisting of proteins with different diffusion coefficients. Commonly used diffusion based mixing approaches in microfluidics are not ideal for this, because their readout is dominated by the already dissociated fast diffusing species. In contrast, our microfluidic device utilizes a different, fast and homogeneous mixing approach independent of diffusion. Fast mixing and a long measurement channel enable us to measure dissociation between timescales of ms to min. Due to the nature of the confocal single molecule method, we can also follow conformational changes during dissociation. The function of this device is first shown by measuring structural propertiesand dissociation rates of heat shock protein 90 (Hsp90) dimers. The full potential is then demonstrated by measuring the fast dissociation of the much smaller model client protein Δ131Δ and the slow dissociation of the cochaperone Sba1 from Hsp90.