Intracellular protein breakdown in thermophilic and mesophilic fungi.
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ABSTRACT:
SUBMITTER: Miller HM
PROVIDER: S-EPMC1168487 | biostudies-other | 1974 Nov
REPOSITORIES: biostudies-other
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Project description:Most proteins from thermophiles or hyperthermophiles are intrinsically thermostable. However, though Methanobacterium thermoautotrophicum ΔH is a thermophilic archaeon with an optimal growth temperature of 65 °C, Mth10b, an atypical member the Sac10b protein family from M. thermoautotrophicum ΔH, seems not intrinsically thermostable. In this work, to clarify the molecular mechanism of Mth10b remaining stable under its physiological conditions, the thermodynamic properties of Mth10b were studied through equilibrium unfolding experiments performed at pH 7.0 monitored by circular dichroism (CD) spectra in detail. Our work demonstrated that Mth10b is not intrinsically thermostable and that due to the masking effect upon the large numbers of destabilizing electrostatic repulsions resulting from the extremely uneven distribution of charged residues over the surface of Mth10b, salt can contribute to the thermostability of Mth10b greatly. Considering that the intracellular salt concentration is high to 0.7 M, we concluded that salt is the key extrinsic factor to Mth10b remaining stable under its physiological conditions. In other word, without salt, 'thermophilic' protein Mth10b is just a mesophilic one.
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Project description:BACKGROUND: Thermophilic proteins sustain themselves and function at higher temperatures. Despite their structural and functional similarities with their mesophilic homologues, they show enhanced stability. Various comparative studies at genomic, protein sequence and structure levels, and experimental works highlight the different factors and dominant interacting forces contributing to this increased stability. METHODS: In this comparative structure based study, we have used interaction energies between amino acids, to generate structure networks called as Protein Energy Networks (PENs). These PENs are used to compute network, sub-graph, and node specific parameters. These parameters are then compared between the thermophile-mesophile homologues. RESULTS: The results show an increased number of clusters and low energy cliques in thermophiles as the main contributing factors for their enhanced stability. Further more, we see an increase in the number of hubs in thermophiles. We also observe no community of electrostatic cliques forming in PENs. CONCLUSION: In this study we were able to take an energy based network approach, to identify the factors responsible for enhanced stability of thermophiles, by comparative analysis. We were able to point out that the sub-graph parameters are the prominent contributing factors. The thermophiles have a better-packed hydrophobic core. We have also discussed how thermophiles, although increasing stability through higher connectivity retains conformational flexibility, from a cliques and communities perspective.
| S-EPMC3009521 | biostudies-literature
Project description:BackgroundThere is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts.ResultsWe have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures.ConclusionsCarefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.
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Project description:The mechanisms for the control of ribosomal protein synthesis have been characterized in detail in Eukarya and in Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and MvaL12) of the mesophilic Methanococcus vannielii has been extensively investigated. As in Bacteria, regulation takes place at the level of translation. The regulator protein MvaL1 binds preferentially to its binding site on the 23S rRNA, and, when in excess, binds to the regulatory target site on its mRNA and thus inhibits translation of all three cistrons of the operon. The regulatory binding site on the mRNA, a structural mimic of the respective binding site on the 23S rRNA, is located within the structural gene about 30 nucleotides downstream of the ATG start codon. MvaL1 blocks a step before or at the formation of the first peptide bond of MvaL1. Here we demonstrate that a similar regulatory mechanism exists in the thermophilic M. thermolithotrophicus and M. jannaschii. The L1 gene is cotranscribed together with the L10 and L11 gene, in all genera of the Euryarchaeota branch of the Archaea studied so far. A potential regulatory L1 binding site located within the structural gene, as in Methanococcus, was found in Methanobacterium thermoautotrophicum and in Pyrococcus horikoshii. In contrast, in Archaeoglobus fulgidus a typical L1 binding site is located in the untranslated leader of the L1 gene as described for the halophilic Archaea. In Sulfolobus, a member of the Crenarchaeota, the L1 gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12). A previously suggested regulatory L1 target site located within the L11 structural gene could not be confirmed as an L1 binding site.
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Project description:The evolutionary adaptations of thermophilic water-soluble proteins required for maintaining stability at high temperature have been extensively investigated. Little is known about the adaptations in membrane proteins, however. Here, we compare many properties of mesophilic and thermophilic membrane protein structures, including side-chain burial, packing, hydrogen bonding, transmembrane kinks, loop lengths, hydrophobicity, and other sequence features. Most of these properties are quite similar between mesophiles and thermophiles although we observe a slight increase in side-chain burial and possibly a slight decrease in the frequency of transmembrane kinks in thermophilic membrane protein structures. The most striking difference is the increased hydrophobicity of thermophilic transmembrane helices, possibly reflecting more stringent hydrophobicity requirements for membrane partitioning at high temperature. In agreement with prior work examining transmembrane sequences, we find that thermophiles have an increase in small residues (Gly, Ala, Ser, and Val) and a strong suppression of Cys. We also find a relative dearth of most strongly polar residues (Asp, Asn, Glu, Gln, and Arg). These results suggest that in thermophiles, there is significant evolutionary pressure to offload destabilizing polar amino acids, to decrease the entropy cost of side chain burial, and to eliminate thermally sensitive amino acids.
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Project description:BACKGROUND: Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity. RESULTS: When expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50?°C and thermal in-activation half-life (t1/2) at 50?°C for 47.6 min, compared to 47?°C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96?°C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates. CONCLUSIONS: Domain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.
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Project description:Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PP(i)-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus and Thermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcales and Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from Methanococcus jannaschii was functionally expressed in Escherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order Thermococcales, the M. jannaschii ADP-PFK has an extremely low K(m) for fructose 6-phosphate (9.6 microM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.
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