No abstract

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We find that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be degraded. These misfolded states can persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, allowing them to bypass the proteostasis machinery and processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. These results explain how proteins misfold into soluble, non-functional conformations that bypass cellular quality controls, and indicate that, unexpectedly, this is a wide-spread cellular phenomenon that can lead to reduced protein function across the cytosolic proteome. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

The specific activity of enzymes can be altered over long time scales in cells by synonymous mutations, which change an mRNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we resolve this issue by applying multiscale modeling to three E. coli enzymes - type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B, and dihydrofolate reductase. This modeling involves coarse-grained simulations of protein synthesis and post-translational behavior, all-atom simulations as a test of robustness, and QM/MM calculations to characterize function. We first demonstrate that our model accurately predicts experimentally measured changes in specific activity due to synonymous mutations. Then, we show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. These long-lived states exhibit reduced catalytic activity, as demonstrated by their increased activation energies for the reactions they carry out. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. The localized misfolding involves noncovalent lasso entanglements - a topology in which the protein backbone forms a loop closed by noncovalent native contacts which is then threaded by another portion of the protein. Such entanglements are often kinetic traps, as they can require a large proportion of the protein to unfold, which is energetically unfavorable, before they disentangle and attain the native state. The near-native-like structures of these misfolded states allow them to bypass the proteostasis machinery and remain soluble, as they exhibit similar hydrophobic surface areas as the native state. These entangled structures persist in all-atom simulations as well, indicating that these conclusions are independent of model resolution. Thus, synonymous mutations cause shifts in the co- and post-translational structural ensemble of proteins, whose altered subpopulations lead to long-term changes in the specific activities of some enzymes. The formation of entangled subpopulations is therefore a mechanism through which changes in translation elongation rate alter ensemble-averaged specific activities, which can ultimately affect the efficiency of biochemical pathways and phenotypic traits.

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.