Archaeal Hsp14 drives substrate shuttling between small heat shock proteins and thermosome: insights into a novel substrate transfer pathway.

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form.

A chaperonin as protein nanoreactor for atom-transfer radical polymerization.

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N-isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein-catalyst conjugate only yielded polymers with PDIs above 1.84.

Flexible interwoven termini determine the thermal stability of thermosomes.

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-ß), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-ß is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αß is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-ß revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.

Weak intra-ring allosteric communications of the archaeal chaperonin thermosome revealed by normal mode analysis.

Chaperonins are molecular machines that use ATP-driven cycles to assist misfolded substrate proteins to reach the native state. During the functional cycle, these machines adopt distinct nucleotide-dependent conformational states, which reflect large-scale allosteric changes in individual subunits. Distinct allosteric kinetics has been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL, undergo concerted subunit motions within each ring, whereas archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. We study these distinct mechanisms through a comparative normal mode analysis of monomer and double-ring structures of the archaeal chaperonin thermosome and GroEL. We find that thermosome monomers of each type exhibit common low-frequency behavior of normal modes. The observed distinct higher-frequency modes are attributed to functional specialization of these subunit types. The thermosome double-ring structure has larger contribution from higher-frequency modes, as it is found in the GroEL case. We find that long-range intersubunit correlation of amino-acid pairs is weaker in the thermosome ring than in GroEL. Overall, our results indicate that distinct allosteric behavior of the two chaperonin classes originates from different wiring of individual subunits as well as of the intersubunit communications.

Self-reporting materials: protein-mediated visual indication of damage in a bulk polymer.

Damage self-reporting materials are able to indicate the presence of microscopic damaged regions by easy to detect signals, such as fluorescence. Therefore, these smart materials can reduce the risk of catastrophic failure of load-bearing components, e.g., in aerospace and construction applications. We highlight here our proof-of-concept paper and we present some additional data, which shows that proteins can be used as mechanophores in solid polymeric materials. Macroscopic mechanical forces were transferred from the polymer to the embedded proteins. The biomolecules act as molecular strain sensor, giving the material the desired self-reporting property. Poly(ethylene glycol) and poly(acrylamide) (PAAm) networks were doped with small amounts of thermsosome (THS), a protein cage from the family of chaperonins, that encapsulated a pair of fluorescent proteins. THS acts as a scaffold which brings the two fluorescent proteins into distance suitable for fluorescence resonance energy transfer (FRET). Moreover, THS can be distorted by mechanic forces so that the distance between the fluorescent proteins changes, leading to a change in FRET efficiency. Using the brittle PAAm as a model system, we were able to visualize microcracks in the polymers by FRET microscopy and by fluorescence lifetime imaging. THS also stabilizes the encapsulated guest proteins against thermal denaturation, increasing their half-live at 70 degrees C by a factor of 2.3.

Insights into chaperonin function from studies on archaeal thermosomes.

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.

Crystal structure of group II chaperonin in the open state.

Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpnß) in the open state from Acidianus tengchongensis. There is a ∼30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnß were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5(T).

Recombinant thermosomes from the Acidianus tengchongensis strain S5(T) were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnalpha or rATcpnbeta) and hetero-oligomers (rATcpnalphabeta). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnalpha homo-oligomer was shown to possess 8-fold symmetry while both rATcpnbeta and rATcpnalphabeta oligomers adopted 9-fold symmetry. rATcpnalphabeta oligomers were shown to contain the alpha and beta subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 degrees C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 degrees C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnbeta sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.

Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei.

Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.

Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins.

Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 beta-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor.