class="head no_bottom_margin" id="sec1title">IntroductionTo function properly in the cell, a globular protein chain typically must remain folded into a specific conformation or set of conformations known as its native state. A primary determinant of how a globular protein folds is its amino acid sequence, which fixes the pattern of internal and external forces that act on the polypeptide chain in the aqueous environment. When a protein cannot reach or cannot maintain its native conformation in the cell, it is considered to be in a misfolded state, which is often accompanied by a loss of cellular function. Misfolded proteins also exhibit a marked tendency to associate non-specifically and sometimes form potentially cytotoxic aggregates if left to accumulate in the crowded intracellular environment (). A large range of globular proteins have been shown to form amyloid fibrils under partially denaturing conditions (). Mutations or stochastic processes that lead to protein misfolding and/or aggregation have been implicated in proteinopathies such as Huntington's, Alzheimer's, Parkinson's, and the prion-based Creutzfeldt-Jakob disease, emphasizing a need to better understand the cellular response to protein misfolding in the context of the physical driving forces that govern how an amino acid sequence can reach its native structure ().The protein quality control (PQC) machinery consists of the cellular pathways linked to protein folding and misfolding. Chaperone proteins, a key component of PQC systems, can either assist a misfolded protein in refolding or target incorrect conformations for destruction through the ubiquitin proteasome pathway or autophagy (). Despite the prevalence of PQC machinery across organisms, many aspects of the system are not well understood. Misfolded proteins are able to reach a wide variety of different non-native states, and the PQC must be able to recognize this diverse group of conformations either to assist refolding or to target them for destruction before the misfolded polypeptide can detrimentally affect the cell. Hsp70, a pleiotropic heat-shock-induced chaperone conserved from bacteria to eukaryotes, has been shown to recognize exposed hydrophobic sites, particularly short (∼5–7 residues) hydrophobic sequences flanked by positively charged amino acids (). The eukaryotic chaperonin TRiC, on the other hand, has eight distinct subunits, which are each capable of recognizing distinct motifs in a variety of substrates, with mutations in different subunits leading to different cellular phenotypes (). Because the outcome of the PQC triage decision must ultimately depend on the structure of a protein, investigating the role of small conformational perturbations in the sensitivity of a misfolded protein to the QC machinery can indicate how the fate of a PQC substrate is modulated for typical substrates.Marginally stable proteins, which can misfold easily and exist at a tipping point between stable conformations and PQC-targeted misfolded variants, have been used as an experimental mechanism to explore PQC substrate recognition and subsequent refolding and degradation pathways. The human von Hippel-Lindau (VHL) protein is one such example that is particularly susceptible to incorrect folding. This model misfolding protein forms part of an E3 ubiquitin ligase complex that targets molecules like HIF-1α for degradation, and has been cataloged in depth because hundreds of mutant forms have been linked to cancer pathways in humans (). The first ∼60 residues of VHL remain disordered in the native state; however, the 213-residue protein as a whole must traverse a distinct folding pathway in vivo, including interacting with chaperones such as Hsp70 and TRiC and binding with its cofactors elongin B and elongin C, to achieve a state resistant to cellular degradation (). For TRiC, two short motifs in the VHL sequence (Box 1 and Box 2) have been shown to be necessary and sufficient for TRiC binding to VHL in yeast (). When folded correctly in complex with its cofactors, the non-disordered region adopts a well-defined tertiary structure; however, the protein adopts a molten globule state without its binding partners in vitro that consists of a partially collapsed state with some secondary structure but no tertiary structure (). This molten globule state indicates that VHL has difficulty achieving its native state without interactions with other proteins. Perturbations to the system, including mutations to VHL, often lead to a misfolded or otherwise non-functional version of the protein in vivo (). When VHL is introduced into non-native systems like Saccharomyces cerevisiae or Escherichia coli, where it does not exist naturally, the protein cannot achieve a biologically stable state and in yeast is quickly degraded by the cell (). Since VHL is a protein with a typical state that is poised between adequate folding and being targeted for destruction in yeast, it is ideal for use as a probe of how different folds (or misfolded variants) can lead to diverse outcomes through PQC pathways.One of the persistent difficulties in understanding the physical mechanisms of protein misfolding and subsequent PQC interactions is that almost by definition, misfolded proteins are not amenable to conventional methods of structural characterization. Protein chains that adopt many different conformations cannot be crystallized easily, and aggregation-prone proteins are difficult to solubilize for in vitro characterization. Thus, in examining the effects of different mutations on a marginally stable protein like VHL, a computational model that could give insight into the resulting structural changes could offer a new and much needed perspective on the connection between sequence, structure, and recognition by PQC machinery for a large number of sequences. Recently, we developed a phenomenological model to predict tertiary structural information from sequence alone in globular proteins, which has shown promise as a method of computationally exploring the allowed conformational space of fluctuating protein folds (). The burial trace is computed by minimizing an energy function consisting of the hydropathies of each residue and the stretching between neighbor amino acids, subject to steric constraints. The calculation generally takes less than a second to run for short sequences, and adding noise to the parameters of the system can generate an ensemble of amino acid burial patterns for a given protein sequence, which can be used to investigate the variability in structures that a protein can adopt. The rapidity of this model in determining structural information makes it an excellent candidate for probing large numbers of potential mutations of marginally stable proteins to understand PQC response to different conformations in silico and to guide in vivo experiments. This analysis could also shed light on a possible functional role for marginal stability, which may enable sensitive modulation of expression through qualitative transitions in conformational state.To investigate the link between the underlying biophysics of protein folding and the PQC fate of a model misfolded protein, the burial mode model was used to investigate the folding characteristics of the human VHL tumor suppressor protein. Burial traces were calculated to predict exposed residues for the lowest energy conformations of different mutations of VHL, 20 of which were generated experimentally and tested for their degradation properties in vivo. One of these mutations had markedly and consistently higher levels of VHL present at steady state. Through the use of burial mode analysis, the structural basis of its enhanced ability to persist in the cell was characterized. Our findings confirm that VHL sits on a structural tipping point in sequence space, where a single mutation can lead to a qualitative shift in folding stability, which leads to an altered quality control outcome. Not only do these results highlight the power of a new computational model in gaining elusive information about the structure of intrinsically disordered proteins, they also raise the possibility that such proteins may generally be poised to exhibit strong sensitivity to mutation in vivo, where small perturbations can lead to large differences in the amount of folded protein that survives PQC supervision.
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