Role of nonspecific interactions in molecular chaperones through model-based bioinformatics

Abstract

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein.

Figure 1

The model results of 528 E. coli proteins. The y axis is the predicted folding free-energy perturbation from the open or trans GroEL complex. A negative number indicates stabilization. The x axis indicates the perturbation from the closed or cis GroEL-GroES complex. Most hypotheses of the GroEL-GroES complex action predict that the closed form should be the most stabilizing, which is indeed observed for the majority (98%) of the proteins (seen above the dashed line).

Figure 2

The folding free-energy perturbation from the GroEL trans or open form. The contribution to the folding free-energy perturbation from the change in internal energy is plotted on the y axis. The internal energy is nearly always positive, indicating all proteins are energetically destabilized in the closed form. The entropy or confinement contribution to the folding free-energy perturbation is shown in the x axis. It is negative for most of the proteins. (Points below the dashed lines) These are stabilized more by confinement than they are destabilized by internal energy.

Figure 3

The residue fractions for the open or trans GroEL conformation (N = 394) and the closed or cis GroEL-GroES complex (N = 119). The key feature here is which residues change. There is a higher fraction of charged residues in the closed form; the open form does have more hydrophobic residues, specifically leucine, isoleucine, valine, and methionine.

Figure 4

The folding free-energy perturbation from the GroEL-GroES cis or closed form. The contribution to the folding free-energy perturbation from the change in internal energy is plotted on the y axis. The internal energy is nearly always negative, indicating nearly all proteins are energetically stabilized in the closed form. The entropy or confinement contribution to the folding free-energy perturbation is shown in the x axis. It is negative for all of the protein. (Points above the dashed lines) These points are stabilized more by confinement than the internal energy, which is rare except for the larger proteins. Note that proteins that cannot fit into the GroEL-GroES complex whereas folded are not included in this analysis.

Figure 5

Extreme values for the model as the residue fractions are changed to single components, where the fraction is 1 for one residue type and 0 for all others. The median folding free-energy perturbation as predicted by the model from GroEL-GroES on 528 E. coli proteins is shown on the y axis. The x axis is the single residue type, which is maximal. For example, the A indicates that only alanine is present on the surface of the GroEL-GroES and the bar height is the median folding free energy for E. coli proteins if only alanines were present in GroEL-GroES. The plot shows that the isoleucine destabilizes proteins the most, along with other hydrophobic residues as expected. Aspartic acid is the most stabilizing residue, followed by asparagine, glutamic acid, and lysine. Cysteine is not expected to be stabilizing or destabilizing due to its unique disulfide bonding.