Protein folding often competes with intermolecular aggregation, which in most cases irreversibly impairs protein function, as exemplified by the formation of inclusion bodies. Although it has been empirically determined that some proteins tend to aggregate, the relationship between the protein aggregation propensities and the primary sequences remains poorly understood. Here, we individually synthesized the entire ensemble of Escherichia coli proteins by using an in vitro reconstituted translation system and analyzed the aggregation propensities. Because the reconstituted translation system is chaperone-free, we could evaluate the inherent aggregation propensities of thousands of proteins in a translation-coupled manner. A histogram of the solubilities, based on data from 3,173 translated proteins, revealed a clear bimodal distribution, indicating that the aggregation propensities are not evenly distributed across a continuum. Instead, the proteins can be categorized into 2 groups, soluble and aggregation-prone proteins. The aggregation propensity is most prominently correlated with the structural classification of proteins, implying that the prediction of aggregation propensity requires structural information about the protein.

Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. A dataset that evaluates which chaperones are effective for aggregation-prone proteins would provide an invaluable resource not only for understanding the roles of chaperones, but also for broader applications in protein science and engineering. Therefore, we comprehensively evaluated the effects of the major Escherichia coli chaperones, trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ∼800 aggregation-prone cytosolic E. coli proteins, using a reconstituted chaperone-free translation system. Statistical analyses revealed the robustness and the intriguing properties of chaperones. The DnaK and GroEL systems drastically increased the solubilities of hundreds of proteins with weak biases, whereas trigger factor had only a marginal effect on solubility. The combined addition of the chaperones was effective for a subset of proteins that were not rescued by any single chaperone system, supporting the synergistic effect of these chaperones. The resource, which is accessible via a public database, can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.

Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome‐wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only ∼60% of Class III substrates are bona fide obligate GroE substrates in vivo . The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation‐prone folds. Notably, ∼70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution.

The interaction of Hsp104 with yeast prion fibers made of Sup35NM, a prion-inducing domain of Sup35, was tested. When fluorescently labeled Hsp104 was added to the preformed fibers, individual fibers were fluorescently decorated uniformly along the fiber length. However, the density of fluorescence differed from one fiber to another, indicating the presence of subspecies of Sup35NM fibers. The time course of fiber formation from monomer Sup35NM was delayed by Hsp104. Hsp104-mediated fragmentation of fibers was tested using bead-tethered fibers. In contrast with the recent report (Shorter, J., and Lindquist, S. (2004) Science 304, 1793–1797), Hsp104 alone was unable to sever the fibers. Yeast cell lysate or the Hsp104-deficient cell lysate plus Hsp104 caused ATP-dependent, guanidine hydrochloride-sensitive fragmentation of the fibers. Thus, in our experimental setup, Hsp104 plus other factor(s) in the yeast cytosol are required for severing yeast prion fiber. The reason of discrepancy from the above report is unknown but is possibly caused by different conformational subspecies of prion fibers.