Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

Abstract

Subpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long time scale changes in protein structure and function can persist in cells because some proteins non-native states can bypass components of the proteostasis machinery.

A Proteome-Wide Map of Chaperone-Assisted Protein Refolding in the Cytosol

“…A second possibility is that chaperones can identify these states but ultimately cannot repair them, targeting them for degradation. The first scenario has recently been investigated by O’Brien and co-workers (Halder et al, 2021), who found computationally that E. coli isochorismate synthase (EntC), enolase (Eno), Galactitol-1-phosphate dehydrogenase (GatD), MetK, and purine nucleoside phosphorylase (DeoD) are prone to form near-native entangled states that bypass GroEL. In agreement, our study identifies Eno and DeoD as chaperone nonrefolders (as for the others: EntC had too low coverage for inclusion; MetK is GroEL-fastidious; GatD was not detected in intrinsic refolding experiments but fully refolded in all chaperone experiments, supporting previous work showing its misfolded states rapidly aggregate (Kerner et al, 2005)).…”

A Newly Identified Class of Protein Misfolding in All-atom Folding Simulations Consistent with Limited Proteolysis Mass Spectrometry

“…Only a small number of mechanisms intrinsic to a protein's primary structure are known to cause monomeric protein misfolding (Table 1). Recently, high-throughput, coarse-grained simulations of protein synthesis and folding of the E. coli proteome have suggested there exists an additional widespread mechanism of misfolding [1][2][3] . Proteins can populate off-pathway misfolded states that involve a change in entanglement 1 .…”

“…The misfolded states observed in the aforementioned simulations involved either the gain of a non-native entanglement (i.e., the formation of an entanglement that is not present in the native ensemble, Table S1) or the loss of a native entanglement (i.e., an entanglement present in the native state fails to form, Table S1) [1][2][3] . This newly predicted class of misfolding offers an explanation for the decades old observations that non-functional protein molecules can persist for long-time scales in the presence of chaperones and not rapidly aggregate nor be degraded [7][8][9] .…”

“…Several mechanisms intrinsic to a protein's primary structure are known to cause monomeric protein misfolding (Table 1). Recently, high-throughput, coarse-grained simulations of protein synthesis and folding of the E. coli proteome have suggested there exists an additional widespread mechanism of misfolding [1][2][3] : proteins can populate off-pathway misfolded states that involve a change in non-covalent lasso entanglements 1,2 . This type of entanglement is defined by the presence of two structural components: a loop formed by a protein backbone segment closed by a non-covalent native contact, and another protein segment that is threaded through and around this loop [4][5][6] in some cases multiple times (Figs.…”

“…S1) or the loss of a native entanglement (i.e., an entanglement present in the native state fails to form, Table S1 and Fig. S1) [1][2][3] . This newly predicted class of misfolding offers an explanation for the decades-old observations that non-functional (or less functional) protein molecules can persist for long timescales without aggregating, and in the presence of chaperones neither get repaired nor degraded [7][8][9] .…”