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

ABSTRACT: 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

SUBMITTER: Hellenkamp B 

PROVIDER: S-EPMC6526119 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature