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PubMed2005

Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress.

Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.

Endoplasmic Reticulum↗
PubMed2006

Crystal structure of the sugar binding domain of the archaeal transcriptional regulator TrmB.

TrmB is an alpha-glucoside-sensing transcriptional regulator controlling two operons encoding maltose/trehalose and maltodextrin ABC transporters of Pyrococcus furiosus. The crystal structure of an N-terminal truncated derivative of TrmB (amino acids 2-109 deleted; TrmB(delta2-109)) was solved at 1.5 A resolution. This protein has lost its DNA binding domain but has retained its sugar recognition site. The structure represents a novel sugar-binding fold. TrmB(delta2-109) bound maltose, glucose, sucrose, and maltotriose, exhibiting Kd values of 6.8, 25, 34, and 160 microM, respectively. TrmB(delta2-109) behaved as a monomer in dilute buffer solution in contrast to the full-length protein, which is a dimer. Co-crystallization with bound maltose identified a binding site involving seven amino acid residues: Ser229, Asn305, Gly320, Met321, Val324, Ile325, and Glu326. Six of these residues interact with the nonreducing glucosyl residue of maltose. The nonreducing glucosyl residue is shared by all substrates bound to TrmB, suggesting it as a common recognition motif.

Amino Acid Motifs↗
PubMed2004

Domain mapping of the Rad51 paralog protein complexes.

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1-75 interacts with the C-terminus and linker of Rad51C, residues 79-376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4-77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77-328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes.

Amino Acid Sequence↗
PubMed2001

Functional interactions of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase delta.

BACKGROUND: By the total genome sequencing of several archaeal organisms, it has been confirmed that many archaeal proteins related to genetic information systems, including DNA replication, transcription and translation, have similar sequences to those of eukaryotes. In eukaryotic DNA replication, proliferating cell nuclear antigen (PCNA) works in clamping DNA polymerases on the DNA template and accomplishes a processive DNA synthesis. Archaea encode PCNA homologues in their genomes and Pyrococcus furiosus PCNA (PfuPCNA) stimulates the DNA synthesizing activities of the DNA polymerases, Pol I and Pol II, in this organism. RESULTS: We have demonstrated that PfuPCNA interacts functionally with calf thymus DNA polymerase delta (Pol delta) and stimulates its activity. Moreover, human replication factor C (RFC) enhances the PfuPCNA-dependent DNA synthesis activity of Pol delta, indicating that human RFC works as the clamp loader for PfuPCNA. These results showed that the three-dimensional structures of archaral PCNA and RFC are actually similar enough to their eukaryotic counterparts to allow a molecular substitution between the two biological domains, albeit at a lower efficiency. CONCLUSIONS: We found that the archaeal molecule interacts functionally with the eukaryotic members in the DNA replication process. This finding supports the idea that studies on the DNA replication mechanism of archaeal organisms will provide many important clues for understanding of the intricate molecular recognition that is inherent to the DNA replication machinery in Eukarya.

Animals↗
PubMed1998

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.

Acids↗
PubMed2000

The orientation of DNA in an archaeal transcription initiation complex.

RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase-promoter interface. The largest component of this archaeal polymerase is split into two subunits, A' and A", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.

Archaeal Proteins↗
PubMed2001

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols.

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.

Base Sequence↗
PubMed2002

Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation.

We discovered a thermostable enzyme from the archaeon Pyrococcus furiosus (Pfu), which increases yields of PCR product amplified with Pfu DNA polymerase. A high molecular mass (>250 kDa) complex with PCR-enhancing activity was purified from Pfu extracts. The complex is a multimer of two discrete proteins, P45 and P50, with significant similarity to bacterial dCTP deaminase/dUTPase and DNA flavoprotein, respectively. When tested in PCR, only recombinant P45 exhibited enhancing activity. P45 was shown to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate. Pfu dUTPase improves the yield of products amplified with Pfu DNA polymerase by preventing dUTP incorporation and subsequent inhibition of the polymerase by dU-containing DNA. dUTP was found to accumulate during PCR through dCTP deamination and to limit the efficiency of PCRs carried out with archaeal DNA polymerases. In the absence of dUTP inhibition, the combination of cloned Pfu DNA polymerase and Pfu dUTPase (PfuTurbo DNA polymerase) can amplify longer targets in higher yield than Taq DNA polymerase. In vivo, archaeal dUTPases may play an essential role in preventing dUTP incorporation and inhibition of DNA synthesis by family B DNA polymerases.

DNA, Archaeal↗
PubMed2006

[Molecular cloning and expression of extremely thermostable and acid-stable amylase gene in Pichia pastoris].

The gene encoding a extremely thermostable and acid-stable alpha-Amylase was amplified by PCR using hyperthermophilic archaebacterium pyrococcus furiosus genomic DNA as template. Then the gene was cloned into the vector of pPIC9K. The recombinant vector pPIC9K-amy was then transformed into E. coli DH5alpha strain. Sequencing test showed that the a-amylase gene cloned consisted of 1305 base pairs and the mature protein encoded by the gene consisted of 435 amino acids. The recombinant vector was transformed into chromosome of methylotrophic yeast Pichia pastoris GS115 strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant a-Amylase was expressed and excreted out of the cells. The expression of the recombinant alpha-amylase was strictly induced by methanol. As induction time increased, the activity of amylase per milliliter medium went up accordingly. After 7 days induction, the activity of the amylase reached the max. The recombinant alpha-amylase exhibited maximal activity at 90 to approximately 100 degrees C and at pHranging from 4.5 to 5.0. The enzyme is so thermostable that after disposed at 100 degrees C for 5 hours over 60% of activity was retained.

Bacterial Proteins↗
PubMed2003

Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability.

Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.

Amino Acid Sequence↗
PubMed2004

Structural features of a hyperthermostable endo-beta-1,3-glucanase in solution and adsorbed on "invisible" particles.

Conformational characteristics and the adsorption behavior of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133 degrees C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.

Adsorption↗
PubMed2006

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

Calorimetry, Differential Scanning↗
PubMed2002

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases.

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).

Aminopeptidases↗
PubMed2006

Protein-protein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors.

Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.

Archaeal Proteins↗
PubMed2004

Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation.

The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C. This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data.

Amino Acid Substitution↗
PubMed1998

Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene.

Adenylosuccinate synthetase (PurA) catalyzes the first step in the de novo AMP synthesis and has been extensively studied in both Bacteria and Eukarya. We cloned the purA gene from the hyperthermophilic archaeon, Pyrococcus furiosus. The gene appears to be individually transcribed and encodes a protein of 339 amino acids. The amino acid sequence comparison with other archael PurAs found from recent genome analyses indicated that two deletions, one central and the other C-terminal, are a common feature of archaeal PurAs. None of the 21 PurA homologues analyzed from Eukarya and Bacteria exhibited this feature. Amino acid sequences of PurAs in Archaea showed 64% average identities which were significantly higher than the 50% and 55% calculated for Bacteria and Eukarya, respectively. Several residues conserved in PurAs of both Eukarya and Bacteria and shown to be of catalytic importance are missing in the archaeal PurAs. Phylogenetic analysis using PurA as the marker grouped life into 3 domains, hence it was consistent with results derived from 16-18S ribosomal RNA sequences. The topology within the three domains, in general, portrayed the hitherto accepted evolutionary relationship among the organisms utilized. PurA can, thus, serve as an additional marker to evaluate phylogenetic inferences drawn from sequence data from rRNA and other conserved genes. The presence of two unique deletions in both euryarchaeal and crenarchaeal PurAs, but not in those of Bacteria and Eukarya, is a strong evidence confirming the common lineage of these two subdomains of Archaea.

Adenylosuccinate Synthase↗
PubMed2003

Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2.

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.

Adenosine Triphosphatases↗
PubMed2003

Hydrogen bonds in rubredoxins from mesophilic and hyperthermophilic organisms.

The extent and strength of the hydrogen bond networks in rubredoxins from the hyperthermophile Pyrococcus furiosus (PfRd), and its mesophilic analogue Clostridium pasteurianum (CpRd), are examined and compared using NMR spectroscopy. NMR parameters examined in this study include through-hydrogen bond (h3)J(NC)(') scalar couplings and (1)H, (13)C, and (15)N chemical shifts, as well as covalent (1)J(NH) and (1)J(NC)(') scalar couplings. These parameters have allowed the characterization in solution of 12 hydrogen bonds in each protein. Despite a 83% sequence homology and a low RMSD for the backbone heavy atoms (0.648 A) in the crystalline state, subtle, but definite, changes have been identified in the detailed hydrogen-bonding patterns. CpRd shows an increased number of hydrogen bonds in the triple-stranded beta-sheet and an additional hydrogen bond in the multiple-turn segment including residues 14-32. On the other hand, PfRd exhibits an overall strengthening of N-H...O=C hydrogen bonds in the loops involved at the metal binding site as well as evidence for an additional NH...S(Cys) hydrogen bond involving the alanine residue 44. These data, as well as temperature dependence of the NMR parameters, suggest that the particular NMR hydrogen bond pattern found in the hyperthermophile rubredoxin leads to an increased stabilization at the metal binding pocket. It seems to result from a subtle redistribution of hydrogen-bonding interactions between the triple-stranded beta-sheet and the actual metal binding site.

Clostridium↗