Kinetics of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing and Fluorescence Stoichiometry release_ytzu3jtgyzglbafecitqis65be

by Björn Hellenkamp, Johann Thurn, Martina Stadlmeier, Thorsten Hugel

Published in Journal of Physical Chemistry B by American Chemical Society (ACS).

2018   Volume 122, Issue 49, p11554-11560

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 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.
In text/plain format

Archived Files and Locations

application/pdf  1.3 MB
file_yxukyimipndyhhjmnewojhmw3i
europepmc.org (repository)
web.archive.org (webarchive)
Read Archived PDF
Preserved and Accessible
Type  article-journal
Stage   published
Date   2018-10-11
Language   en ?
Journal Metadata
Not in DOAJ
In Keepers Registry
ISSN-L:  1520-5207
Work Entity
access all versions, variants, and formats of this works (eg, pre-prints)
Catalog Record
Revision: 5e64a330-a6af-4d48-8613-5f1dc866a7d5
API URL: JSON