Cooperative Assembly of Hsp70 Subdomain Clusters

Biochemistry
Maya A WrightTuomas P J Knowles

Abstract

Many molecular chaperones exist as oligomeric complexes in their functional states, yet the physical determinants underlying such self-assembly behavior, as well as the role of oligomerization in the activity of molecular chaperones in inhibiting protein aggregation, have proven to be difficult to define. Here, we demonstrate direct measurements under native conditions of the changes in the average oligomer populations of a chaperone system as a function of concentration and time and thus determine the thermodynamic and kinetic parameters governing the self-assembly process. We access this self-assembly behavior in real time under native-like conditions by monitoring the changes in the micrometer-scale diffusion of the different complexes in time and space using a microfluidic platform. Using this approach, we find that the oligomerization mechanism of the Hsp70 subdomain occurs in a cooperative manner and involves structural constraints that limit the size of the species formed beyond the limits imposed by mass balance. These results illustrate the ability of microfluidic methods to probe polydisperse protein self-assembly in real time in solution and to shed light on the nature and dynamics of oligomerization processes.

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Citations

Apr 9, 2020·Biophysics Reviews·William E ArterTuomas P J Knowles
Jul 27, 2021·EMBO Molecular Medicine·Marc EmmeneggerAdriano Aguzzi
Oct 16, 2021·Nature Communications·Matthias M SchneiderTuomas P J Knowles

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Methods Mentioned

BETA
protein folding
size exclusion chromatography
Sizing
MDS

Software Mentioned

scipy
MDS
Python

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