Regioselective photocyclodimerization of 2-anthracenecarboxylic acid through ATP hydrolysis-driven conformational change using simulation prediction-designed GroEL mutant.

GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroELMEL, in which the 307‒309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroELMEL-mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent.

Application of image-recognition techniques to automated micronucleus detection in the in vitro micronucleus assay.

BACKGROUND: An in vitro micronucleus assay is a standard genotoxicity test. Although the technique and interpretation of the results are simple, manual counting of the total and micronucleus-containing cells in a microscopic field is tedious. To address this issue, several systems have been developed for quick and efficient micronucleus counting, including flow cytometry and automated detection based on specialized software and detection systems that analyze images. RESULTS: Here, we present a simple and effective method for automated micronucleus counting using image recognition technology. Our process involves separating the RGB channels in a color micrograph of cells stained with acridine orange. The cell nuclei and micronuclei were detected by scaling the G image, whereas the cytoplasm was recognized from a composite image of the R and G images. Finally, we identified cells with overlapping cytoplasm and micronuclei as micronucleated cells, and the application displayed the number of micronucleated cells and the total number of cells. Our method yielded results that were comparable to manually measured values. CONCLUSIONS: Our micronucleus detection (MN/cell detection software) system can accurately detect the total number of cells and micronucleus-forming cells in microscopic images with the same level of precision as achieved through manual counting. The accuracy of micronucleus numbers depends on the cell staining conditions; however, the software has options by which users can easily manually optimize parameters such as threshold, denoise, and binary to achieve the best results. The optimization process is easy to handle and requires less effort, making it an efficient way to obtain accurate results.

In vivo client proteins of the chaperonin GroEL-GroES provide insight into the role of chaperones in protein evolution.

Protein folding is often hampered by intermolecular protein aggregation, which can be prevented by a variety of chaperones in the cell. Bacterial chaperonin GroEL is a ring-shaped chaperone that forms complexes with its cochaperonin GroES, creating central cavities to accommodate client proteins (also referred as substrate proteins) for folding. GroEL and GroES (GroE) are the only indispensable chaperones for bacterial viability, except for some species of Mollicutes such as Ureaplasma. To understand the role of chaperonins in the cell, one important goal of GroEL research is to identify a group of obligate GroEL/GroES clients. Recent advances revealed hundreds of in vivo GroE interactors and obligate chaperonin-dependent clients. This review summarizes the progress on the in vivo GroE client repertoire and its features, mainly for Escherichia coli GroE. Finally, we discuss the implications of the GroE clients for the chaperone-mediated buffering of protein folding and their influences on protein evolution.

TEM and STEM-EDS evaluation of metal nanoparticle encapsulation in GroEL/GroES complexes according to the reaction mechanism of chaperonin.

Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.

Asp-52 in combination with Asp-398 plays a critical role in ATP hydrolysis of chaperonin GroEL.

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼ 150 h (∼ 6 days), providing a good model to characterize the football-shaped complex.

Crystal structure of a symmetric football-shaped GroEL:GroES2-ATP14 complex determined at 3.8Å reveals rearrangement between two GroEL rings.

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES2 complex (the "football"-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES2-ATP14 complex from Escherichia coli at 3.8Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the "bullet" complex). However, the two GroES-bound GroEL rings form a modified interface by an ~7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.

Yeast prion protein New1 can break Sup35 amyloid fibrils into fragments in an ATP-dependent manner.

We report a new type of interaction between two yeast prion proteins, Sup35 and New1. New1 consists of an N-terminal prion region (New1N) and a C-terminal region homologous to a translation elongation factor with two ATP-binding motifs. Amyloid formation of the Sup35 prion region (Sup35NM) was accelerated by a small amount of sonicated New1N amyloid (New1N-seeds) to produce Sup35NM[New1] amyloid. New1N amyloid formation was accelerated by Sup35NM[New1]-seeds but not by spontaneously generated Sup35NM-seeds, indicating that the structural features of the New1N amyloid were transmitted via the Sup35NM amyloid. Surprisingly, full-length New1 broke the Sup35NM amyloid fibrils in an ATP-dependent manner. This activity of New1 was independent from Hsp104. It was lost by a mutation in the second ATP-binding motif, by the truncation of the N-terminal prion region of New1 and by the pre-incubation of New1 with New1N-seeds. When New1 was overproduced in yeast [PSI(+)] cells carrying GFP-fused Sup35NM, diverse morphological changes in fluorescent foci occurred. Thus, New1 potentially has a regulatory role in prion state in yeast, working independently of the Hsp104 system.

Cryo-EM structure of the native GroEL-GroES complex from thermus thermophilus encapsulating substrate inside the cavity.

The chaperonin GroEL interacts with various proteins, leading them to adopt their correct conformations with the aid of GroES and ATP. The actual mechanism is still being debated. In this study, by use of cryo-electron microscopy, we determined the solution structure of the Thermus thermophilus GroEL-GroES complex encapsulating its substrate proteins. We observed the averaged density of substrate proteins in the center of the GroEL-GroES cavity. The position of the averaged substrate density in the cavity suggested a repulsive interaction between a majority of the substrate proteins and the interior wall of the cavity, which is suitable for substrate release. In addition, we observed a distortion of the cis-GroEL ring, especially at the position near the substrate, which indicated that the interaction between the encapsulated proteins and the GroEL ring results in an adjustment in the cavity's shape to accommodate the substrate.

Revisiting the GroEL-GroES reaction cycle via the symmetric intermediate implied by novel aspects of the GroEL(D398A) mutant.

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325-338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the "football"-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination.

Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL.

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers.

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.

Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity.

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.

Crystallization of the chaperonin GroEL-GroES complex from Thermus thermophilus HB8.

The chaperonin GroEL-GroES (GroEL/ES) complex from a thermophilic eubacteria, Thermus thermophilus HB8, has been purified and crystallized. The GroEL/ES complex is known to be composed of 14 identical GroEL subunits (58 kDa) and seven identical GroES subunits (11 kDa). The GroEL/ES complex crystals belong to the triclinic space group P1, with unit-cell parameters a = 140.4, b = 156.4, c = 273.1 A, alpha = 82.9, beta = 85.4, gamma = 68.5 degrees. The crystal asymmetric unit contains two molecules (MW = 885 kDa). One data set to 3.0 A resolution, with 383 652 independent observations (89.3% complete) and an R(merge) of 0.08, has been collected from a single crystal. A molecular-replacement solution was obtained using the structure of the GroEL/ES complex from Escherichia coli as a search model.

Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.