Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

Exploring the influence of hyperthermophilic protein Ssh10b on the stability and conformation of RNA by molecular dynamics simulation

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which binds RNA in vivo as a physiological substrate, and it has been postulated to play a key role in chromosomal organization in Archaea. Even though the crystal structure of Ssh10b‐RNA was resolved successively by X‐ray diffraction (Protein Data Bank [PDB] code: 3WBM), the detailed dynamic characteristics of Ssh10b‐RNA are still unclear. In this study, molecular dynamics (MDs) simulations at 6 temperatures (300, 350, 375, 400, 450, and 500 K) and molecular mechanics Generalized‐Born surface area (MM‐GB/SA) free energy calculations were performed to investigate the mechanism of how Ssh10b protects and stabilizes RNA. The simulation results indicate that RNA is stabilized by Ssh10b when the temperature rises up to 375 K. RNA is found to undergo conformational transition between A‐RNA and A′‐RNA when Ssh10b binds to RNA at 3 different temperatures (300, 350, and 375 K). Salt bridges, hydrogen bonds and hydrophobic interactions are observed, and some residues have significant impact on the structural stability of the complex. This study increases our understanding of the dynamics and interaction mechanism of hyperthermophilic proteins and RNA at the atomic level, and offers a model for studying the structural biology of hyperthermophilic proteins and RNA.     

Ssh10b2与其同源蛋白Ssh10b在细胞含量及固定DNA超螺旋的能力上存在明显差异

极端嗜热古菌———芝田硫化叶菌(Sulfolobus shibatae)基因组含一对亲缘关系较远的同源基因,ssh10b和ssh10b2。这对同源基因编码的蛋白(Ssh10b和Ssh10b2)属于古菌Sac10b DNA结合蛋白家族。关于Ssh10b以及与其极为相似的硫矿硫化叶菌(S.solfataricus)Sso10b、嗜酸热硫化叶菌(S.acidocaldarius)Sac10b蛋白已有较多研究,推测这些蛋白可能在染色体组织和包装、DNA重组、基因表达调控等方面起作用。克隆并在大肠杆菌中表达了ssh10b2基因,纯化了重组Ssh10b2蛋白。免疫印迹定量分析表明,ssh10b2在芝田硫化叶菌中有表达,但其细胞含量仅相当于Ssh10b的约十分之一。重组Ssh10b2对双链DNA的亲和力低于Ssh10b。此外,Ssh10b2和Ssh10b在与双链DNA结合时表现出相似的凝胶阻滞模式。有意思的是,Ssh10b2固定DNA负超螺旋的能力明显低于Ssh10b。这些结果提示,Ssh10b和Ssh10b2可能具有不同的生理作用。     

Ssh10b2 differs from its paralogue Ssh10b in cellular abundance and the ability to constrain DNA supercoils]

The ssh10b and ssh10b2 genes, a pair of distantly related paralogues in Sulfolobus shibatae, encode members of the Sac10b DNA binding protein family in thermophilic archaea. It has been shown previously that Ssh10b exists in abundance in S. shibatae and is capable of constraining negative DNA supercoils, properties that are consistent with a speculated architectural role for the protein in chromosomal organization. In this study, the ssh10b2 gene was cloned and expressed in Escherichia coli, and the recombinant Ssh10b2 protein was purified to apparent homogeneity. Immunoblotting analysis using a specific anti - Ssh10b2 antibody showed that ssh10b2 was expressed in S. shibatae, but the cellular level of Ssh10b2 was only - 10% of that of Ssh10b. Recombinant Ssh10b2 was capable of interacting with both double-stranded and single-stranded DNA. The affinity of the protein for double-stranded DNA was higher than that reported for Ssh10b. The Ssh10b2 and Ssh10b proteins appeared to generate similar gel shift patterns on duplex DNA fragments. However, unlike Ssh10b, Ssh10b2 was unable to constrain DNA supercoils. These data suggest that Ssh10b2 does not serve as a general architectural factor in DNA compaction and organization in S. shibatae.     

Favorable contribution of the C-terminal residue K97 to the stability of a hyperthermophilic archaeal [P62A]Ssh10b

The role of residue K97 at the C-terminal end of archaeal [P62A]Ssh10b in the hyperthermostability of the protein is investigated using three K97-mutant variants: K97E-, K97A-, and ΔK97-mutant [P62A]Ssh10b. The thermal- and GdmHCl-induced denaturation of the three mutant variants has been monitored by circular dichroism. The results reveal that the K97E mutation leads to a stronger destabilization effect than the K97A mutation by disturbing the electrostatic interaction of the salt-bridge D63-K97 and drawing an unfavorable charge-charge repulsive interaction into the structure. However, ΔK97-[P62A]Ssh10b shows much lower stability than K97E- and K97A-mutant [P62A]Ssh10b. Analysis suggests that residue K97 at the C-terminal end makes the favorable contributions to the stability of hyperthermophilic [P62A]Ssh10b not only by the favorable electrostatic interactions with residues in close vicinity but also through maintaining the side chain packing of the surrounding residues in the C-terminal area of the protein.     

Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo

Proteins of the Sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as DNA-binding proteins. Based on their in vitro DNA-binding properties, these proteins are thought to be involved in chromosomal organization or DNA repair/recombination. We show that Ssh10b, a member of the Sac10b family from Sulfolobus shibatae, bound with similar affinities to double-stranded DNA, single-stranded DNA and RNA in vitro. However, the protein was exclusively bound to RNA in S. shibatae cells, as revealed by in vivo UV cross-linking and co-immunoprecipitation. Ribosomal RNAs were among the RNA species co-immunoprecipitated with Ssh10b. Consistent with this observation, Ssh10b was co-purified with ribosomes under low salt conditions. Furthermore, we demonstrate by UV-cross-linking hybridization that, when the cells were irradiated with UV, Ssh10b became cross-linked to 16S, 23S rRNAs and mRNAs. Our data indicate that RNA is the physiological binding target of the Sac10b family.     

Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔH^cal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.     

Disulfide bridges are not involved in penicillin-binding protein 1b dimerization in Escherichia coli

PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers.     

Salt bridges in the miniature viral channel Kcv are important for function

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K⁺ channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K⁺ ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing.     

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2

We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.     

Biochemical and Structural Insights into RNA Binding by Ssh10b,a Member of the Highly Conserved Sac10b Protein Family in Archaea

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.     

Crystal structure of the hyperthermophilic archaeal DNA-binding protein Sso10b2 at a resolution of 1.85 Angstroms

The crystal structure of a small, basic DNA binding protein, Sso10b2, from the thermoacidophilic archaeon Sulfolobus solfataricus was determined by the Zn multiwavelength anomalous diffraction method and refined to 1.85 A resolution. The 89-amino-acid protein adopts a betaalphabetaalphabetabeta topology. The structure is similar to that of Sso10b1 (also called Alba) from the same organism. However, Sso10b2 contains an arginine-rich loop RDRRR motif, which may play an important role in nucleic acid binding. There are two independent Sso10b2 proteins in the asymmetric unit, and a plausible stable dimer could be deduced from the crystal structure. Topology comparison revealed that Sso10b2 is similar to several RNA-binding proteins, including IF3-C, YhhP, and DNase I. Models of the Sso10b2 dimer bound to either B-DNA or A-DNA have been constructed.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

A stabilizing alpha/beta-hydrophobic core greatly contributes to hyperthermostability of archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.     

Glycosylation increases the thermostability of human aquaporin 10 protein

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.     

Extremely halophilic archaeal communities are resilient to short-term entombment in halite

Some haloarchaea avoid the harsh conditions present in evaporating brines by entombment in brine inclusions within forming halite crystals, where a subset of haloarchaea survives over geological time. However, shifts in the community structure of halite-entombed archaeal communities remain poorly understood. Therefore, we analysed archaeal communities from in situ hypersaline brines collected from Trapani saltern (Sicily) and their successional changes in brines versus laboratory-grown halite over 21 weeks, using high-throughput sequencing. Haloarchaea were dominant, comprising >95% of the archaeal community. Unexpectedly, the OTU richness of the communities after 21 weeks was indistinguishable from the parent brine and overall archaeal abundance in halite showed no clear temporal trends. Furthermore, the duration of entombment was less important than the parent brine from which the halite derived in determining the community composition and relative abundances of most genera in halite-entombed communities. These results show that halite-entombed archaeal communities are resilient to entombment durations of up to 21 weeks, and that entombment in halite may be an effective survival strategy for near complete communities of haloarchaea. Additionally, the dominance of ‘halite specialists’ observed in ancient halite must occur over periods of years, rather than months, hinting at long-term successional dynamics in this environment.     

DNA topological change in the hyperthermophilic archaeon Pyrococcus abyssi exposed to low temperature

Abstract Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.     

Two disulphide bridges are present in juvenile hormone binding protein from Galleria mellonella

The hemolymph juvenile hormone binding protein (JHBP) from Galleria mellonella contains two disulphide bridges/molecule and no free Cys residues. An alignment of primary structures of other Lepidopteran JHBPs indicates that Cys residues, equivalent to Cys10,17,151,195 in G. mellonella JHBP, maybe involved in -S-S- bridge formation.