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Cooperative Assembly of Hsp70 Subdomain Clusters

Authors: Maya A. Wright, Francesco A. Aprile, Mathias M. J. Bellaiche, Thomas C. T. Michaels, Thomas Müller, Paolo Arosio, Michele Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles

Biochem., 2018, 57, 3641-3649. DOI: 10.1021/acs.biochem.8b00151 

 

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, they 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.

They 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, they 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.

In this paper, Wright et al. use Microfluidic Diffusional Sizing (MDS) to study the oligomerization of SBD641, the substrate binding subdomain of human Hsp70. By revealing the thermodynamic parameters governing the oligomerization, they show structural constraints on the oligomer size are likely determined by specific molecular interaction modes at the interface.

Wright et al Hsp70 abstract figure

 

While it is known that molecular chaperones play a crucial role in assisting protein folding, the authors note that existing analysis techniques are better suited to testing monodisperse solutions of isolated components - so MDS has offered a new avenue for these types of tests.

Measurements of the unlabelled sample were conducted using a Fluidity One instrument in order to verify that the fluorescent label used in the study did not affect the measured hydrodynamic radius.

Read the full paper

 


Instrument: Fluidity One
Therapeutic area: protein oligomerization

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