Constant relative rate of protein evolution and detection of functional diversification among bacterial, archaeal and eukaryotic proteins

Background  

Detection of changes in a protein's evolutionary rate may reveal cases of change in that protein's function. We developed and implemented a simple relative rates test in an attempt to assess the rate constancy of protein evolution and to detect cases of functional diversification between orthologous proteins. The test was performed on clusters of orthologous protein sequences from complete bacterial genomes (Chlamydia trachomatis, C. muridarum and Chlamydophila pneumoniae), complete archaeal genomes (Pyrococcus horikoshii, P. abyssi and P. furiosus) and partially sequenced mammalian genomes (human, mouse and rat).     

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395 m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98°C (maximum growth temperature 102°C), but capable of prolonged survival at 105°C. Optimum growth was at pH 7 (range 5–8) and NaCl concentration 2.4% (range 1%–5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn²⁺-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA–DNA hybridization (<63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp. nov. Received: December 10, 1997 / Accepted: February 4, 1998     

Probing protein electrostatic interactions through temperature/reduction potential profiles

 The change in the equilibrium reduction potentials of the iron-sulfur proteins, Pyrococcus furiosus rubredoxin and P. furiosus ferredoxin, and heme protein, horse cytochrome c, has been calculated as a function of temperature using a numerical solution to the Poisson-Boltzman equation. Working curves for different internal dielectric constants were generated to best reproduce experimental observation. Based on a comparison of the experimental and simulated change in reduction potential with temperature, it is concluded that the dielectric constant of proteins is temperature-dependent and varies from protein to protein. For example, the temperature-dependent reduction potential of cytochrome c can only be simulated using a different temperature-dependent dielectric constant for each oxidation state, but this was not the case for rubredoxin or ferredoxin. The role of changes in ionization states of cytochrome c at alkaline pHs, where the reduction potential is known to be pH-dependent at room temperature, is also discussed in terms of electrostatic interaction energies as a function of temperature. It appears that temperature/reduction potential profiles may provide a direct method for measuring relative changes in internal protein dielectric constants. Received: 29 April 1996 / Accepted: 1 August 1996     

Analysis of the Hyperthermophilic Chitinase from Pyrococcus furiosus: Activity toward Crystalline Chitin

Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the β-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.     

Hyperthermophilic redox chemistry: a re-evaluation

The redox chemistry of Pyrococcus furiosus rubredoxin and ferredoxin has been studied as a function of temperature in direct voltammetry and in EPR monitored bulk titrations. The E_ms of both proteins, measured with direct voltammetry, have a normal (linear) temperature dependence and show no pH dependence. EPR monitoring is not a reliable method to determine the temperature dependence of the E_m: upon rapid freezing the proteins take their conformation corresponding to the freezing point of the solution.     

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.     

Structural insights into the interaction of the Nip7 PUA domain with polyuridine RNA

The conserved protein Nip7 is involved in ribosome biogenesis, being required for proper 27S pre-rRNA processing and 60S ribosome subunit assembly in Saccharomyces cerevisiae. Yeast Nip7p interacts with nucleolar proteins and with the exosome subunit Rrp43p, but its molecular function remains to be determined. Solution of the Pyrococcus abyssi Nip7 (PaNip7) crystal structure revealed a monomeric protein composed by two alpha-beta domains. The N-terminal domain is formed by a five-stranded antiparallel beta-sheet surrounded by three alpha-helices and a 310 helix while the C-terminal, a mixed beta-sheet domain composed by strands beta8 to beta12, one alpha-helix, and a 310 helix, corresponds to the conserved PUA domain (after Pseudo-Uridine synthases and Archaeosine-specific transglycosylases). By combining structural analyses and RNA interaction assays, we assessed the ability of both yeast and archaeal Nip7 orthologues to interact with RNA. Structural alignment of the PaNip7 PUA domain with the RNA-interacting surface of the ArcTGT (archaeosine tRNA-guanine transglycosylase) PUA domain indicated that in the archaeal PUA domain positively charged residues (R151, R152, K155, and K158) are involved in RNA interaction. However, equivalent positions are occupied by mostly hydrophobic residues (A/G160, I161, F164, and A167) in eukaryotic Nip7 orthologues. Both proteins can bind specifically to polyuridine, and RNA interaction requires specific residues of the PUA domain as determined by site-directed mutagenesis. This work provides experimental verification that the PUA domain mediates Nip7 interaction with RNA and reveals that the preference for interaction with polyuridine sequences is conserved in Archaea and eukaryotic Nip7 proteins.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

Re-Annotation of Two Hyperthermophilic Archaea <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis> GE5 and <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> DSM 3638

Pyrococcus abyssi GE5 (P. aby) and Pyrococcus furiosus DSM 3638 (P. fur) are two model hyperthermophilic archaea. However, their annotations in public databases are unsatisfactory. In this article, the two genomes were re-annotated according to the following steps. (i) All “hypothetical genes” in the original annotation were re-identified based on the Z-curve method, and some of them were recognized as non-coding open reading frames (ORFs). Evidence showed that the recognized non-coding ORFs were highly unlikely to encode proteins. (ii) The translation initiation sites (TISs) of all the annotated genes were re-located, and more than 10% of the TISs were shifted to 5′-upstream or 3′-downstream regions. (iii) The functions of the refined “hypothetical genes” were predicted using sequence alignment tools, more than 200 originally annotated “hypothetical genes” in either of the two hyperthermophiles were assigned functions. A large number of these functions have reference support or experimentally characterized homologues. All the refined information will serve as a valuable resource for research on P. aby and P. fur, which may be helpful in the exploration of thermal adaptation mechanisms. The complete re-annotation files of P. aby and P. fur are available at .     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs)

Background  

Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi.     

Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus

Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC–MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles. Received: 29 March 1999 / Accepted: 25 May 1999     

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol⁻¹. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.     

UV and Ethyl Methanesulfonate Effects in Hyperthermophilic Archaea and Isolation of Auxotrophic Mutants of Pyrococcus Strains

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi. Received: 2 May 1996 / Accepted: 3 July 1996     

Crystal structure and nucleic acid‐binding activity of the CRISPR‐associated protein Csx1 of Pyrococcus furiosus

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR‐associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C‐terminal domain of PF1127 has a unique β‐hairpin structure that protrudes out of an α‐helix and contains several positively charged residues. We demonstrate that PF1127 binds double‐stranded DNA and RNA and that this activity requires an intact β‐hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β‐hairpin structure and exhibits no DNA‐binding properties under the same experimental conditions. Notably, the C‐terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N‐terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid‐binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin

Background  

Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions.     

MutS2 Family Protein from Pyrococcus furiosus

MutS2 protein of Pyrococcus furiosus has been cloned and over-expressed. Initial characterization reveals that PfuMutS2 possesses a thermostable ATPase activity and a thermostable, nonspecific DNA binding activity. However, PfuMutS2 does not have any detectable mismatch-specific DNA binding activity. It is the first in vitro characterization of an MutS2 family protein. Received: 23 April 2001 / Accepted: 27 August 2001