Elucidating the novel mechanisms of molecular chaperones by single-molecule technologies.

Molecular chaperones play central roles in sustaining protein homeostasis and preventing protein aggregation. Most studies of these systems have been performed in bulk, providing averaged measurements, though recent single-molecule approaches have provided an in-depth understanding of the molecular mechanisms of their activities and structural rearrangements during substrate recognition. Chaperone activities have been observed to be substrate specific, with some associated with ATP-dependent structural dynamics and others via interactions with co-chaperones. This Review aims to describe the novel mechanisms of molecular chaperones as revealed by single-molecule approaches, and to provide insights into their functioning and its implications for protein homeostasis and human diseases.

Overexpression of chaperones GroEL/ES from Escherichia coli enhances indigo biotransformation production of cytochrome P450 BM3 mutant.

The self-sufficient cytochrome P450 BM3 mutant (A74G/F87V/D168H/L188Q) can serve as a biocatalyst for whole-cell catalysis process of indigo. Nevertheless, the bioconversion yield of indigo is generally low under normal cultivation conditions (37 °C, 250 rpm). In this study, a recombinant E. coli BL21(DE3) strain was constructed to co-express the P450 BM3 mutant gene and GroEL/ES genes to investigate whether GroEL/ES can promote the indigo bioconversion yield in E. coli. The results revealed that the GroEL/ES system could significantly increase the indigo bioconversion yield, and the indigo bioconversion yield of the strain co-expressing P450 BM3 mutant and GroEL/ES was about 21-fold that of the strain only expressing the P450 BM3 mutant. In addition, the P450 BM3 enzyme content and in vitro indigo bioconversion yield were determined to explore the underlying mechanism for the improvement of indigo bioconversion yield. The results revealed that GroEL/ES did not increase indigo bioconversion yield by increasing the content of P450 BM3 enzyme and its enzymatic transformation efficiency. Moreover, GroEL/ES could improve the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP+ ratio. Given that NADPH is an important coenzyme in the catalytic process of indigo, the underlying mechanism for the improvement of indigo bioconversion yield is probably related to an increase in the intracellular NADPH/NADP+ ratio.

Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the GroEL/ES complex, which resulted in noticeably elevated enzyme activity with one of the de-novo-designed promoters of the library. The new toolbox offers a straightforward one-pot cloning approach and is highly modular. As such, the method presented here should be of great interest to any gene co-expression study.

Overexpression and kinetic analysis of Ideonella sakaiensis PETase for polyethylene terephthalate (PET) degradation.

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETaseMut, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETaseMut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L-1 purified soluble IsPETaseMut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L-1 purified soluble NusA-IsPETaseMut, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETaseMut (GroEL/ES) displayed the same catalytic behavior as IsPETaseMut, while the fusion of NusA conferred new enzymatic properties to IsPETaseMut. Although NusA-IsPETaseMut displayed a lower initial hydrolysis capacity than IsPETaseMut, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETaseMut. Taken together, the latter two catalytic properties of NusA-IsPETaseMut are more likely to contribute to the enhanced product release by NusA-IsPETaseMut PET degradation for two weeks.

Multimodal approaches for the improvement of the cellular folding of a recombinant iron regulatory protein in E. coli.

BACKGROUND: During the recombinant protein expression, most heterologous proteins expressed in E. coli cell factories are generated as insoluble and inactive aggregates, which prohibit E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The presence of folding modulators such as exogenous molecular chaperones or osmolytes, as well as process variables such as incubation temperature, inducer concentrations, growth media are all important for cellular folding and are investigated in this study. This study also investigated how the cell's stress response system activates and protects the proteins from aggregation. RESULTS: The cells exposed to osmolytes plus a pre-induction heat shock showed a substantial increase in recombinant aconitase activity when combined with modulation of process conditions. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells. CONCLUSIONS: The recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase production during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP.

Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the process is generally very fast, and we need a suitable read out. In this protocol, we have solved this issue by using a protein that is non-fluorescent in its unfolded state but fluoresces in its native state after folding. The kinetics of refolding can be monitored by following the increase in fluorescence in real time. Previously, this was generally achieved by either monitoring a protein's enzymatic activity or measuring the tryptophan fluorescence, where the signal output depends on well-described enzymatic activity or the frequency of tryptophan residues present in the proteins, respectively. Here, we describe a simple and real-time assay to monitor the refolding of sGFP, a recently described slow-folding mutant of yeGFP (yeast enhanced GFP). We unfold this protein using chemical denaturant and refold in a suitable buffer, monitoring the increase in fluorescence over time. GFP is fluorescent only when correctly folded; thus, using this technique, we can measure the true rate of protein refolding by following the increase in fluorescence over time. Therefore, sGFP can be used as an ideal model to study the in vitro protein folding process. Accordingly, the effects of different conditions and molecules on the protein folding pathway can be efficiently studied using sGFP as a model protein. Graphical abstract: Schematic of the steps involved in the sGFP refolding pathway. Native sGFP is unfolded by chemical denaturation using 6 M GuHCl at 25°C for 1 hour and then refolded in refolding buffer by 100-fold dilution.

Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

[Ligand-Induced Reassembly of GroEL/ES Chaperone In Vitro: Visualization by Electron Microscopy].

The products of the reassembly reaction of tetradecameric two-ring quaternary structure of GroEL chaperonin under the pressure of its heptameric co-chaperonin GroES have been visualized by electron microscopy. It has been shown that one-ring heptameric oligomers of GroEL have been formed at the beginning (after ~5 min) of the reaction, while at the final stage of the reaction (after ~70 min), both one-ring heptamers in complex with one GroES and two-rings tetradecamers in complexes with one (asymmetrical complex) or two (symmetrical complex) GroES heptamers are present. The relationship between the data of light scattering, native electrophoresis, and electron microscopy obtained earlier has been discussed.

Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon.

Role of calcium in the mitigation of heat stress in the cyanobacterium Anabaena PCC 7120.

The effects of exogenously added CaCl2 (0.25mM) on photopigments, photosynthetic O2-evolution, antioxidative enzyme activity, membrane damage, expression of two heat shock genes (groEL and groES) and apoptotic features in Anabaena 7120 under heat stress (45°C) for up to 24h were investigated. Heat stress lowered the level of photopigments; however, Ca2+--supplemented cultures showed a low level reduction in Chl a but induced accumulation of carotenoids and phycocyanin under heat stress. Photosynthetic O2-evolving capacity was maintained at a higher level in cells from Ca2+-supplemented medium. Among the antioxidative enzymes, superoxide dismutase activity was unaffected by the presence or absence of Ca2+ in contrast to increases in catalase, ascorbate peroxidase and glutathione reductase activities in cells grown in Ca2+-supplemented medium. Lower levels of lipid peroxidation were recorded in Anabaena cells grown in Ca2+-supplemented medium in comparison to cells from Ca2+--deprived medium. Target cells grown in Ca2+-deprived medium developed apoptotic features in the early stages of heat shock, while Ca2+ application seemed to interfere with apoptosis because only a few cells showed such features after 24 h of heat exposure, indicating a role for Ca2+ in maintaining cell viability under heat stress. There was also continuous up regulation of two important heat shock genes (groEL and groES) in Ca2+-supplemented cultures, exposed to heat shock, again indicating a role for Ca2+ in stress management.

Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies.

Chitobiase (CHB) is an important enzyme for the production of N-acetyl-D-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58%) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42% CHB in soluble form and the rest (~58%) as inclusion bodies. The percentage of active CHB was enhanced to 71% by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG--0.5 mM, L-arabinose--13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37% could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M L-arginine, 5% glycerol). Using combinatorial approach, 80% of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.

Trigger Factor Reduces the Force Exerted on the Nascent Chain by a Cotranslationally Folding Protein.

Cotranslational protein folding can generate pulling forces on the nascent chain that can affect the instantaneous translation rate and thereby possibly feed back on the folding process. Such feedback would represent a new way of coupling translation and folding, different from coupling based on, for example, codon usage. However, to date, we have carried out the experiments used to measure pulling forces generated by cotranslational protein folding either in reconstituted in vitro translation systems lacking chaperones, in ill-defined cell lysates, or in vivo; hence, the effects of chaperones on force generation by folding are unknown. Here, we have studied the cotranslational folding of dihydrofolate reductase (DHFR) in the absence and in the presence of the chaperones trigger factor (TF) and GroEL/ES. DHFR was tethered to the ribosome via a C-terminal linker of varying length, ending with the SecM translational arrest peptide that serves as an intrinsic force sensor reporting on the force generated on the nascent chain when DHFR folds. We find that DHFR folds into its native structure only when it has emerged fully outside the ribosome and that TF and GroEL alone substantially reduces the force generated on the nascent chain by the folding of DHFR, while GroEL/ES has no effect. TF therefore weakens the possible coupling between cotranslational folding and translation.

Enhanced expression of soluble human papillomavirus L1 through coexpression of molecular chaperonin in Escherichia coli.

The major recombinant capsid protein L1 of human papillomavirus (HPV) is widely used to produce HPV prophylactic vaccines. However, the quality of soluble and active expression of L1 in Escherichia coli was below the required amount. Coexpression with the chaperonin GroEL/ES enhanced L1 expression. Overexpressing GroEL/ES increased the soluble expression level of glutathione S-transferase-fused L1 (GST-L1) by approximately ∼3 fold. The yield of HPV type 16 L1 pentamer (L1-p) was ∼2 fold higher than that in a single expression system after purification through size-exclusion chromatograph. The expression and purification conditions were then optimized. The yield of L1-p was enhanced by ∼5 fold, and those of HPV types 18 and 58 L1-p increased by ∼3 and ∼2 folds, respectively, compared with that in the single expression system. Coexpressing the mono-site mutant HPV16 L1 L469A with GroEL/ES increased L1-p yield by ∼7 fold compared with strains expressing the wild-type L1 gene. L1-p was then characterized using circular dichroism spectra, UV-vis cloud point, dynamic light scattering and transmission electron microscope analyses. Results indicated that the conformation and biological characteristics of L1-p were identical to that of native L1. Hence, overexpressing chaperonin in E. coli can increase the expression level of GST-L1 and L1-p production after purification. This finding may contribute to the development of a platform for prophylactic HPV vaccines.

GroEL-assisted protein folding: does it occur within the chaperonin inner cavity?

The folding of protein molecules in the GroEL inner cavity under the co-chaperonin GroES lid is widely accepted as a crucial event of GroEL-assisted protein folding. This review is focused on the data showing that GroEL-assisted protein folding may proceed out of the complex with the chaperonin. The models of GroEL-assisted protein folding assuming ligand-controlled dissociation of nonnative proteins from the GroEL surface and their folding in the bulk solution are also discussed.