Equatorial Domain Research Articles

Unfolding and disassembly of the chaperonin GroEL occurs via a tetradecameric intermediate with a folded equatorial domain.

The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces. This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea. Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography. Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain. Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain. This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded. Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected. This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly.

Purification of GroEL from an overproducing E. coli strain.

Molecular chaperones are a class of abundant and ubiquitous proteins, which assist and accelerate protein folding in the cell. Classification of these proteins is continually changing owing to advances in our understanding of how these molecules function. One major group, the chaperonins (1), are defined as large, multimeric proteins that assemble into a double ring (or “double-doughnut”) structure. The crystal structure of the chaperonin GroEL from Escherichia coli (2) showed the 14 subunits arranged in two rings of 7 subunits. Each subunit has three distinct domains, the equatorial domain (through which the rings are joined), a hinge region (which facilitates movement), and the apical domain (where the unfolded protein substrate binds). There are many models for the mechanism of action of this molecule (for recent reviews, see 3), however it is generally accepted that the unfolded protein binds to the apical domain and is sequestered inside the cavity of the chaperonin owing to the binding of the cochaperonin, GroES, to the same sites on the apical domains. Release of the protein from the chaperonin occurs after ATP hydrolysis. Chaperonins are broadly further classified into two other groups based on source and sequence comparisons: Type I, which comprises bacterial, mitochondrial, and chloroplast chaperonins, and Type II, which comprises the archaeal and eukaryotic cytoplasmic chaperonins. All chaperonins have a subunit size of approx 60 kDa. GroEL has become the most examined and standard chaperonin in the field of chaperone-mediated protein folding. This chapter describes a purification protocol by which it is possible to obtain pure GroEL from an overproducing strain of E. coli. Several such strains have been produced in many laboratories and new constructs of GroEL, and its homologs can be generated easily using standard molecular biology techniques. Two main properties of the active chaperonin are exploited during its purification, that of its low isoelectric point (pI = 4.7) and large size (14×57 kDa).

Functional communications between the apical and equatorial domains of GroEL through the intermediate domain.

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.

Warm Water Paths in the Equatorial Atlantic as Diagnosed with a General Circulation Model

Abstract Monthly mean velocity fields from a global ocean general circulation model are used to study the main circulation patterns within the upper 1200 m of the equatorial Atlantic. Some recently developed Lagrangian techniques are used to picture and quantify the routes followed in the model by distinct water mass classes, defined by their initial temperature on model transatlantic sections at 10°S and 10°N. The qualitative description in terms of equatorial pathways of this warm component of the so-called global “conveyor belt” is found coherent with the most recent circulation schemes inferred from direct measurements. Diagnostics emphasize the crucial role of the western boundary current system and that of the equatorial subsurface jets in distributing the flow in the equatorial domain, both for northward-flowing and southward-recirculating warm water masses. As the model tracer fields are constrained to remain close to the observed climatology outside the equatorial strip, the circulation calculate...

Single molecular observation of the interaction of GroEL with substrate proteins

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine β-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.

Molecular characterization of the 5′ control region and of two lethal alleles affecting the hsp60 gene in Drosophila melanogaster

The chaperonins are evolutionarily conserved essential cellular proteins that help folding newly synthesized or translocated proteins, spending ATP. We present here the molecular analysis of the hsp60 gene promoter region and of two Drosophila hsp60 ethyl methane sulfonate embryonic lethal alleles that have an identical phenotype. No heat shock element sequences were found in the 5′ region, supporting previous data (Kozlova, T. et al., 1997) which suggests that mitochondrial Drosophila melanogaster HSP60.1 is not heat inducible. By sequencing the lethal allele’s entire open reading frame (ORF), we found a C-T transition in the hsp60 F409 allele that produces a serine to leucine change, apparently distorting the protein equatorial domain structure. No changes were found in the hsp60 G93 ORF. However, an analysis of the heterogeneous nuclear RNA levels showed a reduction of the hsp60 transcript in hsp60 G93 flies as compared to the wild-type. These data suggest that although the defects in the hsp60 gene produced by these alleles are at different levels, both behave as null mutations.

Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase.

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.

A Single Ring Is Sufficient for Productive Chaperonin-Mediated Folding In Vivo

Facilitated protein folding by the double toroidal bacterial chaperonin, GroEL/GroES, proceeds by a “two-stroke engine” mechanism in which an allosteric interaction between the two rings synchronizes the reaction cycle by controlling the binding and release of cochaperonin. Using chimeric chaperonin molecules assembled by fusing equatorial and apical domains derived from GroEL and its mammalian mitochondrial homolog, Hsp60, we show that productive folding by Hsp60 and its cognate cochaperonin, Hsp10, proceeds in vitro and in vivo without the formation of a two-ring structure. This simpler “one-stroke” engine works because Hsp60 has a different mechanism for the release of its cochaperonin cap and bound target protein.

Chemistry of surface sediment along a north–south transect across the equator in the Central Indian Basin: an assessment of biogenic and detrital influences on elemental burial on the seafloor

Nineteen surface sediment samples collected along a north–south transect across the equator in the Central Indian Basin (CIB) were analysed for their major, minor, and rare earth element (REE) composition. In absence of the element-flux data, we demonstrate here the association of various elements and their preferential sinks using inter-elemental associations in sediment exhibiting latitudinal dissimilarity. Biogenic sedimentation north of the equator (1° to 4°N latitudes) is dominated by carbonate ooze, whereas south of the equator (4° to 14°S latitudes) is dominated by siliceous ooze. Carbonate ooze deposition (>60% CaCO 3) above 4400 m and its drastic reduction (to <10%) below 4700-m water depths place the carbonate lysocline and the compensation depth (CCD) in a narrow depth zone of around 300-m thickness in the CIB. The siliceous ooze deposition is evident only below 5050-m water depth. This equatorial biogenic sediment domain is interrupted by a detrital clay zone (0° to 3°S latitudes) probably due to a combined effect of reduced dilution of distal Bengal fan sediment below CCD and focusing of suspended detritus drawn from the Bay of Bengal during vigorous southwest monsoon equatorial circulation. The latitudinal trends in the Element XS (detrital unsupported or non-terrigenous element) composition of the sediment and inter-elemental correlations yield four main carrier phases responsible for burial of several elements; (a) carbonate phase burying almost all Ca and Sr; (b) detrital phase burying the majority of Al, Fe, Mg and all of Ti and Sc; (c) oxide phase burying bulk of Mn,Co,Cu and Ba; and (d) biogenic phase burying the Al XS. Ba exhibits latitudinal similarity and strong positive association ( r=∼0.8 at 99.9% confidence level) with Mn, Co and Cu in both Element bulk and Element XS correlation matrices. The proportion of Ba XS, Mn XS, Co XS and Cu XS is >80% of their respective total content in all the three sediment zones. These observations suggest a close link between Ba and a Mn-oxide phase. On the other hand, nearly overlapping latitudinal trends of Al XS with carbonate in areas north of the equator and opal south of the equator; strong positive correlation of Al XS with opal ( r=+∼0.8), similar ratios between carbonate-free opal and Al XS (∼16) in both biogenic sediments suggest a close link between productivity and Al XS. The Al XS proportion in both biogenic sediment types is around 28% of the total Al suggesting nearly the same level of primary productivity along the transect (4°N to 14°S) which supports the estimates of Berger [Berger, W.H., 1989. Global maps of the ocean productivity. In: W.H. Berger, V.S. Smetacek, G. Wefer (Eds.), Productivity of the Ocean: Present and past. Dahlem workshop reports, Wiley, Chichester, pp. 429–455] and Kabanova [Kabanova, J.G., 1968. Primary production of the northern part of the Indian Ocean. Oceanology, 8: 214–255] (i.e., ∼70 g Cm −2 y −1). The REEs exhibit mutual coherence in the sediment ( r=>+0.7). However, they display, an increasing affinity towards Mn (` r' increases from +0.6 to +0.8) and decreasing affinity towards Ti (` r' decreases from +0.8 to +0.3) with increasing atomic number. Such a subtle but systematic variation within REE group suggests preferential burial of heavy-REE by Mn-oxide phase and light-REE by detrital phase.

ATP Binding Induces Large Conformational Changes in the Apical and Equatorial Domains of the Eukaryotic Chaperonin Containing TCP-1 Complex

The chaperonin-containing TCP-1 complex (CCT) is a heteromeric particle composed of eight different subunits arranged in two back-to-back 8-fold pseudo-symmetric rings. The structural and functional implications of nucleotide binding to the CCT complex was addressed by electron microscopy and image processing. Whereas ADP binding to CCT does not reveal major conformational differences when compared with nucleotide-free CCT, ATP binding induces large conformational changes in the apical and equatorial domains, shifting the latter domains up to 40 degrees (with respect to the inter-ring plane) compared with 10 degrees for nucleotide-free CCT or ADP-CCT. This equatorial ATP-induced shift has no counterpart in GroEL, its prokaryotic homologue, which suggests differences in the folding mechanism for CCT.

Potato leafroll virus binds to the equatorial domain of the aphid endosymbiotic GroEL homolog.

A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.

11] Construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL

The chapter presents a study related to the construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL. A single-ring version of the bacterial chaperonin, GroEL, has been designed using information from the crystal structure of the native double-ring assembly. This molecule, designated SR1, has proved to be functional as a folding chamber, inside which proteins, such as rhodanese and green fluorescent protein (GFP), bound to the ring in nonnative form, can reach native form on addition of GroES and ATP. In particular, the substrate protein folds to the native state within the central channel underneath GroES and remains within this space because the absence of a second GroEL ring prevents the normal ATP mediated signaling that releases GroES. SR1 has been designed by examination of the model of unliganded GroEL at 2.8 Å, derived from the orthorhombic crystal. This reveals a contact surface between the two rings that is composed of both electrostatic and hydrophobic interactions. Each subunit in the homotetradecameric complex forms contacts through the inferior aspect of its equatorial domain with that region of two neighboring subunits in the opposite ring. The essential double-ring nature of GroEL, with critical signaling occurring between its rings during the folding reaction, encouraged the study of double-ring complexes containing defects in only one ring.

Analysis of mutationally altered forms of the Cct6 subunit of the chaperonin from Saccharomyces cerevisiae.

The Cct double-ring chaperonin complex of Saccharomyces cerevisiae is comprised of eight essential subunits, Cct1p-Cct8p, and assists the folding of substrates such as actins and tubulins. Single and multiple amino acid replacements of Cct6p were constructed by oligonucleotide-directed mutagenesis, including changes of charged to alanine residues and uncharged to charged residues. The replacements were targeted, in part, to residues corresponding to functionally critical regions identified in the published crystal structure of the Escherichia coli chaperonin, GroEL. Here, we report the critical hydrophobic residues and clusters of hydrophilic residues in regions corresponding to those from the apical domain of GroEL implicated in peptide binding and peptide release, and certain residues in the putative equatorial domain implicated in subunit-to-subunit interaction. In contrast to their homologous counterparts in Cct2p and Cct1p, the highly conserved putative ATP binding motifs of Cct6p were relatively amenable to mutations. Our data suggest that the entire Cct6p molecule might be essential for assembly of Cct complex and might participate in binding substrates. However, there appeared to exist a functional hierarchy in ATP binding/hydrolysis among Cct subunits, as suggested by the high tolerance of Cct6p to mutations within the putative ATP binding pocket.

The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex.

Chaperonins assist protein folding with the consumption of ATP. They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back. In Escherichia coli, asymmetric intermediates of GroEL are formed with the co-chaperonin GroES and nucleotides bound only to one of the seven-subunit rings (the cis ring) and not to the opposing ring (the trans ring). The structure of the GroEL-GroES-(ADP)7 complex reveals how large en bloc movements of the cis ring's intermediate and apical domains enable bound GroES to stabilize a folding chamber with ADP confined to the cis ring. Elevation and twist of the apical domains double the volume of the central cavity and bury hydrophobic peptide-binding residues in the interface with GroES, as well as between GroEL subunits, leaving a hydrophilic cavity lining that is conducive to protein folding. An inward tilt of the cis equatorial domain causes an outward tilt in the trans ring that opposes the binding of a second GroES. When combined with new functional results, this negative allosteric mechanism suggests a model for an ATP-driven folding cycle that requires a double toroid.

ATP-, K+-dependent heptamer exchange reaction produces hybrids between GroEL and chaperonin from Thermus thermophilus.

Chaperonin from Thermus thermophilus (Tcpn6014.Tcpn107) splits at the plane between two Tcpn607 rings into two parts in a solution containing ATP and K+ (Ishii, N., Taguchi, H., Sasabe, H., and Yoshida, M. (1995) FEBS Lett. 362, 121-125). When Escherichia coli GroEL14 was additionally included in the solution described above, hybrid chaperonins GroEL7.Tcpn607 and GroEL7. Tcpn607.Tcpn107 were formed rapidly (<20 s) at 37 degrees C. The hybrid was also formed from Tcpn6014 and GroEL14 but not from a mutant GroEL14 lacking ATPase activity. The hybrid formation was saturated at approximately 300 microM ATP and approximately 300 mM K+. These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction with Thermus chaperonin under nearly physiological conditions. Similar to parent chaperonins, the isolated hybrid chaperonins exhibited ATPase activity that was susceptible to inhibition by Tcpn107 or GroES7 and mediated folding of other proteins. Once formed, the hybrid chaperonins were stable, and the parent chaperonins were not regenerated from the isolated hybrids under the same conditions in which the hybrids had been formed. Only under conditions in which GroEL in the hybrids was selectively destroyed, such as incubation at 70 degrees C, Thermus chaperonin, but not GroEL14, was regenerated from the hybrid. Therefore, the split reaction may not be an obligatory event repeated in each turnover of the chaperonin functional cycles but an event that occurs only when chaperonin is first exposed to ATP/K+.

Detection of changes in pairwise interactions during allosteric transitions: coupling between local and global conformational changes in GroEL.

A protein engineering approach for detecting and measuring local conformational changes that accompany allosteric transitions in proteins is described. Using this approach, we can identify interactions that are made or broken during allosteric transitions. The method is applied to probe for changes in pairwise interactions in the chaperonin GroEL during its ATP-induced allosteric transitions. Two pairwise interactions are investigated: one between subunits (Asp-41 with Thr-522) and the other within subunits (Glu-409 with Arg-501). We find that the intraring intersubunit interaction between Asp-41 and Thr-522 changes little during the allosteric transitions of GroEL, indicating that the hydrogen bond between these residues is maintained. In contrast, the intrasubunit salt bridge between Glu-409 and Arg-501 becomes significantly weaker during the ATP-induced allosteric transitions of GroEL. Our results are consistent with the electron microscopy observations of an ATP-induced hinge movement of the apical domains relative to the equatorial domains.

GroEL Locked in a Closed Conformation by an Interdomain Cross-link Can Bind ATP and Polypeptide but Cannot Process Further Reaction Steps

It has been believed that when GroEL binds to GroES its apical domain moves upward and outward. To inhibit this "opening" movement, its equatorial and apical domains were cross-linked through a disulfide bond between mutationally introduced cysteine residues at the positions of Asp-83 and Lys-327. To avoid possible undesired cross-linking, we at first prepared a mutant GroEL (GroELNC; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser) in which all cysteine residues in wild-type GroEL were replaced by serine residues. GroELNC was fully functional as a chaperonin. We then introduced the above two point mutations into GroELNC to generate a mutant (GroELAEX; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser and Asp-83 --> Cys, Lys-327 --> Cys). Oxidized GroELAEX, which is locked in a "closed" conformation by an interdomain disulfide bond, can bind 6-7 mol of ATP, which remain bound without hydrolysis. This ATP-bound, oxidized GroELAEX can bind the stably nonnative substrate protein isopropylmalate dehydrogenase, whereas the nucleotide-free oxidized GroELAEX binds it with a weaker affinity. However, oxidized GroELAEX fails to process further reaction steps such as ATP hydrolysis, binding of GroES, dissociation of substrate protein from GroEL, and facilitating protein folding. When disulfide bonds in oxidized GroELAEX are reduced, GroELAEX exerts the ability to process all the reactions just as GroELNC and wild-type GroEL. Indications from these results are: hydrolysis of ATP may require opening movement of the apical domain; GroES binds to an open form of GroEL; and substrate polypeptide is released from GroEL coupled with either ATP hydrolysis or opening movement of the apical domain.

The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching and Movements of Substrate-Binding Domains in GroEL

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements. Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES. Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 Å resolution for GroEL and GroEL–GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP. Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein–binding sites. We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

The 2.4 Å crystal structure of the bacterial chaperonin GroEL complexed with ATPγS

GroEL is a bacterial chaperonin of 14 identical subunits required to help fold newly synthesized proteins. The crystal structure of GroEL with ATP gamma S bound to each subunit shows that ATP binds to a novel pocket, whose primary sequence is highly conserved among chaperonins. Interaction of Mg2+ and ATP involves phosphate oxygens of the alpha-, beta- and gamma-phosphates, which is unique for known structures of nucleotide-binding proteins. Although bound ATP induces modest conformational shifts in the equatorial domain, the stereochemistry that functionally coordinates GroEL's affinity for nucleotides, polypeptide, and GroES remains uncertain.

Numerical modeling of the response of Ceará continental shelf waters to wind stress forcing

Ceara shelf waters response to steady and transicnt wind forcing, from Ponta Cajuais (04°42'S, 37°19.5'W) to Ponta de Itapage (02°50.5'S, 40000'W), was studied using a hydrodynamic numerical model. The finitc difference model was based upon the equations for a barotropic ocean in general form: advective and quadratic surface and bottom stress terms were included. Seasonal differences in the shelf waters response were characterized by simulation of summer and winter forcing. Such experiments wcre conducted for both f and equatorial beta plane domains, without significative differences in the results. Transient shelf waters response were investigated using surface winds from the Fortaleza airport , during the period extending from 14/03 to 25/03n9. Model results were compared to current data collected during the same period; modeled currents were able to roughly describe observed current pattem, but not current intensity.