Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins

Abstract

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.cell-free translation ͉ protein aggregation ͉ protein folding

Conversion of a Chaperonin GroEL-independent Protein into an Obligate Substrate

“…Aggregation Propensities of GroE-dependent UuMetK Mutants-Almost all GroE obligate substrates show strong aggregation propensities, according to the global aggregation analysis under the chaperone-free conditions (4,13). Thus, we investigated the in vitro aggregation properties of representative UuMetK mutants, K215F and K215F/I216F, which were converted to GroE-dependent substrates, by a conventional light scattering assay.…”

“…However, folding often competes with a side reaction, intermolecular aggregate formation, which usually impairs the functions of proteins (2,3). Indeed, a global aggregation analysis of thousands of Escherichia coli proteins, using a reconstituted cellfree translation system, revealed that ϳ30% of proteins are aggregation-prone (4). The cellular milieu, where proteins and other molecules exist in highly crowded conditions, further increases the risk of aggregate formation (5,6).…”

“…The canonical chaperones assist the folding of many proteins by preventing irreversible aggregate formation (3,9,10). In vitro, most of the aggregation-prone proteins, which were revealed by the reconstituted cell-free translation system (4), are rescued by one or more conserved chaperones, such as GroEL/GroES (GroE) 2 or DnaK/DnaJ/GrpE (DnaK system) (11). Overall, the effects of GroE and the DnaK system are similar, but certain fractions of proteins are biased toward either GroE or DnaK, probably reflecting the chaperone specificities (11).…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…The algorithms used to map surface properties (electrostatic potential and hydrophobicity) are based on a protein solubility database of E. coli proteins 51. This study has shown that the algorithm can be implemented in the context of recombinant protein production in a mammalian expression system.…”

“…Sequence and structural predictions of protein solubility were obtained from computational work based on comparison with the solubility database of all E. coli proteins (eSOL) which contains the solubility distribution of 3173 E. coli proteins produced in a cell‐free expression system 51. It was found that the experimental solubility values (eSOL) were, on average, inversely correlated with size of calculated largest positive electrostatic potential patch 37.…”