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.
Highlights
We find that the oligomerization mechanism of the Hsp[70] 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
SBD641 consists of the hydrophobic linker, the substrate binding subdomain itself, and the helical lid, which are the regions that have been found to be critical for oligomerization in full-length Hsp70.14 We show that the rapid nature of the microfluidic sizing measurements under native solution conditions makes this approach important for quantitative studies of the kinetics as well as the thermodynamics of oligomerization processes
We found the radius of unlabeled SBD641 at 0.3 μM [10 mM sodium phosphate buffer] to be 1.89 ± 0.25 nm, in agreement with our labeled construct (Figure 2, blue data point)
Summary
Molecular chaperones play a crucial role in vivo in assisting in protein folding and in regulating the formation of aberrant protein assemblies, including in particular those implicated in the onset and progression of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases.[1−9] Despite their important cellular roles, defining the microscopic mechanisms by which molecular chaperones can protect against pathogenic aggregation remains a substantial challenge to study as a result of the polydispersity of the systems involved.[10−13] many molecular chaperones, including the 70 kDa heat shock proteins (Hsp70s) and small heat shock proteins such as αB-crystallin, are known to have the propensity to self-assemble into various oligomeric forms, and it has been thought that this serves as a control mechanism for storage of chaperones when they are not exerting their cellular functions.[12,14−29] Evaluating the intrinsic dynamics of molecular chaperones in the presence and absence of their interaction partners is a key target in biophysical studies to begin to define the specific oligomeric species responsible for the suppression of protein aggregation. A number of conventional biophysical techniques are currently available for probing the quaternary structure and oligomeric populations of proteins; these approaches, are typically more reliable for monodisperse solutions of isolated components.[14,30−32] many of the techniques involve the interaction of the protein of interest with a surface or a matrix, which may influence the self-assembly behavior It has recently become apparent, that microfluidic techniques offer a fruitful avenue for the characterization of the dynamics, interactions, and physical properties of biomolecules in solution.[8,9,16,33−39] These approaches include methods for the rapid sizing of biological complexes in solution by monitoring the spatiotemporal evolution of their diffusion through a microfluidic channel.[39−41] In this work, we have applied microfluidic diffusional sizing (MDS) to the study of the oligomerization of SBD641, the substrate binding subdomain (residues 384−641) of human Hsp[70]. We demonstrate a robust method for determining the dissociation constant, oligomerization free energy, and association and dissociation rate constants of the self-assembly reaction and show that the assembly process is guided by specific structural constraints and is qualitatively different from unconstrained polymerization processes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
