Macromolecular Crowding Extended to a Heptameric System: The Co-chaperonin Protein 10

Abstract

Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.

Macromolecular Crowding In Vitro , In Vivo , and In Between

“…It follows that an experiment designed to follow the concerted formation of a large oligomer would be expected to be much more sensitive to excluded volume effects than one designed to follow the formation of a homo- or heterodimer. Large changes in self-association equilibria were indeed observed in studies of the effect of crowding on the concerted formation of hexamers of ClpB 34 , heptamers of cp10 32 and decamers of bovine pancreatic trypsin inhibitor 86 .…”

Effects of Crowding on the Stability of a Surface-Tethered Biopolymer: An Experimental Study of Folding in a Highly Crowded Regime

“…While this increase in stability presumably arises due to the same electrostatically and sterically driven excluded volume effects postulated to underlie solution-phase crowding, 17 its magnitude is rather greater than the effects typically seen in solution. 18,28 This difference may arise in part due the high local concentrations of DNA that can be achieved in the surface-bound state, which approach 100 mg/mL within one contour length (the 15 nm length of the unfolded DNA chain) of the surface. This said, the stability of proteins (in the dilute regime) typically increases by only ∼1 kJ/mol with the addition of ∼100 mg/mL of highly soluble, nonbiomolecular crowding agents, such as polyvinylpyrrolidone 29 or dextran, 30,31 suggesting that additional mechanisms may be playing a role in our results.…”

Influence of crowded cellular environments on protein folding, binding, and oligomerization: Biological consequences and potentials of atomistic modeling

“…As expected, stabilization much greater than that for binary complexes of single‐domain proteins is observed. For example, using magnetic relaxation dispersion, Snoussi and Halle [76] found a 17000‐fold increase in the association constant for BPTI decamer by 230 g/l of dextran 10 K. Similarly, Aguilar et al [77], based on tryptophan fluorescence, found that 300 g/l Ficoll resulted in a 300‐fold increase in the association constant for heptamer formation of mitochondrial cochaperonin protein 10.…”