Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.     

GroEL/S Overexpression Helps to Purge Deleterious Mutations and Reduce Genetic Diversity during Adaptive Protein Evolution

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way.     

Effects of Topology and Sequence in Protein Folding Linked via Conformational Fluctuations

Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-β, whereas the folding of more local, all-α proteins typically exhibits a stronger sequence dependence. Here, we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3α, 4β + α, and β-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free-energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2000 sequence variants representing all possible hydrophobic or polar single- and double-point mutations. From an analysis of the subset of stability-neutral mutations, we find that folding is perturbed in a topology-dependent manner, with the β-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the β-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.     

Galectin-1 as a fusion partner for the production of soluble and folded human β-1,4-galactosyltransferase-T7 in E. coli

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, β-1,4-galactosyltransferase-7 (β4Gal-T7), in E. coli. The enzyme β4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, β4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6× His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-β4Gal-T7 fusion protein, the unique protease cleavage site allows the protein β4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded β4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.     

A systematic survey of in vivo obligate chaperonin‐dependent substrates

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.     

GroE prevents the accumulation of early folding intermediates of pre-beta-lactamase without changing the folding pathway.

In folding studies of pre-beta-lactamase in the presence of GroE, we investigated the pH dependence of the folding reaction. Two critical intermediates in the folding pathway were defined kinetically. I1 is an early folding intermediate recognized by GroE; the misfolding of I1 leads to aggregation, and this is prevented by GroE. A second intermediate I2 is released from GroE after ATP hydrolysis. Its pH-dependent misfolding to a nonnative form, which is not an aggregate, is not prevented by GroE. From these results, a model is proposed, in which the crucial role of GroE consists of allowing the change from I1 to I2 to take place in the complex. Fluorescence spectra of the pre-beta-lactamase complexed to GroE are very similar to those of the native state. The pathway of pre-beta-lactamase folding is not changed by GroE as evidenced by the same half-time and pH dependence of the folding reaction. GroE probably recognizes the signal sequence and some portion of the mature protein since mature beta-lactamase does not interact with GroE even under conditions of slow folding.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

GroEL–GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL–ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL–GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL–ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL–ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.     

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.     

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.     

High salt-induced conversion of Escherichia coli GroEL into a fully functional thermophilic chaperonin

The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.     

Bacteriophage-encoded cochaperonins can substitute for Escherichia coli's essential GroES protein

The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10–15% of E. coli’s unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli’s essential, housekeeping proteins.     

Evolution of Escherichia coli for Growth at High Temperatures

Evolution depends on the acquisition of genomic mutations that increase cellular fitness. Here, we evolved Escherichia coli MG1655 cells to grow at extreme temperatures. We obtained a maximum growth temperature of 48.5 °C, which was not increased further upon continuous cultivation at this temperature for >600 generations. Despite a permanently induced heat shock response in thermoresistant cells, only exquisitely high GroEL/GroES levels are essential for growth at 48.5 °C. They depend on the presence of lysyl-tRNA-synthetase, LysU, because deletion of lysU rendered thermoresistant cells thermosensitive. Our data suggest that GroEL/GroES are especially required for the folding of mutated proteins generated during evolution. GroEL/GroES therefore appear as mediators of evolution of extremely heat-resistant E. coli cells.     

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

A Disulfide Bond-Containing Alkaline Phosphatase Triggers a BdbC-Dependent Secretion Stress Response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1–2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations.     

Filamentous morphology in GroE-depleted Escherichia coli induced by impaired folding of FtsE

The chaperonin GroE (GroEL and the cochaperonin GroES) is the only chaperone system that is essential for the viability of Escherichia coli. It is known that GroE-depleted cells exhibit a filamentous morphology, suggesting that GroE is required for the folding of proteins involved in cell division. Although previous studies, including proteome-wide analyses of GroE substrates, have suggested several targets of GroE in cell division, there is no direct in vivo evidence to identify which substrates exhibit obligate dependence on GroE for folding. Among the candidate substrates, we found that prior excess production of FtsE, a protein engaged in cell division, completely suppressed the filamentation of GroE-depleted E. coli. The GroE depletion led to a drastic decrease in FtsE, and the cells exhibited a known phenotype associated with impaired FtsE function. In the GroE-depleted filamentous cells, the localizations of FtsA and ZipA, both of which assemble with the FtsZ septal ring before FtsE, were normal, whereas FtsX, the interaction partner of FtsE, and FtsQ, which is recruited after FtsE, did not localize to the ring, suggesting that the decrease in FtsE is a cause of the filamentous morphology. Finally, a reconstituted cell-free translation system revealed that the folding of newly translated FtsE was stringently dependent on GroEL/GroES. Based on these findings, we concluded that FtsE is a target substrate of the GroE system in E. coli cell division.     

Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE.Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.     

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

A distinct three-dimensional shape of rRNA inside the ribosome is required for the peptidyl transfer activity of its peptidyltransferase center (PTC). In contrast, even the in vitro transcribed PTC RNA interacts with unfolded protein(s) at about five sites to let them attain their native states. We found that the same set of conserved nucleotides in the PTC interact identically with nascent and chemically unfolded proteins in vivo and in vitro, respectively. The time course of this interaction, difficult to follow in vivo, was observed in vitro. It suggested nucleation of folding of cytosolic globular proteins vectorially from hydrophilic N to hydrophobic C termini, consistent with our discovery of a regular arrangement of cumulative hydrophobic indices of the peptide segments of cytosolic proteins from N to C termini. Based on this observation, we propose a model here for the nucleation of folding of the nascent protein chain by the PTC.