Xieported here are the first results of a qcantitative thermodynamic st-dy involvixg a series of ... more Xieported here are the first results of a qcantitative thermodynamic st-dy involvixg a series of proteins isolated from the anaerobic sulfur-reducixg archaebacterium Pyrococcus furiosus which grows near submrize geotherml vents on the deep ocean floor. The proteins isolated and investigated here are the two non-henie iron proteins rubredoxin (Kid 5400), and ferredoxin (Mw 7 5 0 0) , and the er-zyine glutamate dehydrogeriase GDH (Xw 270000). The enzyme is inactive at 4OoC and fully activated at 3 0 O C. Independent of their particular primary structure ax! independent of the molecular weight (Mw) all three proteins investigated unfold at exactly the same temperature (113OC) The experimentally observed transition temperature Tm coincides with the extrapolated conversion temperature T* (Privalov, Privalov & Gill, Murphy and Gill, lreire and Murphy, 1-4) of 112OC. It is readily accepted that at this temperature the hydrophobic interactions will cease to contribute to the overall stability of the ordered secondary and/or tertiary structure causing the core of any globular protein to unfold to the denatured state. Thus 1 1 3 O C seems to be the threshold temperature for sustantation of life on earth.
Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temper... more Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122°C. The C-terminal 15-25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif (528 EKEKEKEGEK5 37) proved to be a key domain for stabilization at or near 100°C. Mutations ''tuned'' the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca 2+ , especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca 2+. The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.
The NADP +-specific glutamate dehydrogenase from Escherichia coli has been purified to electropho... more The NADP +-specific glutamate dehydrogenase from Escherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase from E. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The Km values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.
Page 1. Chapter 6 Key Enzymes in the Primary Nitrogen Metabolism of a Hyperthermophile Frank T. R... more Page 1. Chapter 6 Key Enzymes in the Primary Nitrogen Metabolism of a Hyperthermophile Frank T. Robb1, Yaeko Masuchi1, Jae-Bum Park2, and Michael WW Adams2 1Center for Marine Biotechnology, University of Maryland ...
Recombinant Protein Expression: Prokaryotic Hosts and Cell-Free Systems, 2021
Expression of heterologous genes in Escherichia coli is a routine technology for recombinant prot... more Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.
We describe circular polarization as a remote sensing diagnostic of chiral signatures which may b... more We describe circular polarization as a remote sensing diagnostic of chiral signatures which may be applied to Mars. The remarkable phenomenon of homochirality provides a unique biosignature which can be amenable to remote sensing through circular polarization spectroscopy. The natural tendency of microbes to congregate in close knit communities would be beneficial for such a survey. Observations of selected areas of the Mars surface could reveal chiral signatures and hence explore the possibility of extant or preserved biological material. We describe a new instrumental technique that may enable observations of this form.
The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic ar... more The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level ...
All of the important catalytic tasks in living cells are carried out by proteins folded into conf... more All of the important catalytic tasks in living cells are carried out by proteins folded into conformations that are essential for the biological function of the cell.
This report was prepared as an account of work sponsored by an agency of the United States Govern... more This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employes, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors exprcssed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
F1000 - Post-publication peer review of the biomedical literature, 2009
The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encode... more The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origens. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and-200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
F1000 - Post-publication peer review of the biomedical literature, 2008
We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pres... more We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO 2. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (؊34‰ to؊27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<؊12‰), and the kinetic isotope effect at 122°C and 40 MPa was ؊9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane. carbon isotope fractionation ͉ deep-sea hydrothermal vent ͉ hyperthermophile ͉ methanogenesis ͉ piezophilic
Xieported here are the first results of a qcantitative thermodynamic st-dy involvixg a series of ... more Xieported here are the first results of a qcantitative thermodynamic st-dy involvixg a series of proteins isolated from the anaerobic sulfur-reducixg archaebacterium Pyrococcus furiosus which grows near submrize geotherml vents on the deep ocean floor. The proteins isolated and investigated here are the two non-henie iron proteins rubredoxin (Kid 5400), and ferredoxin (Mw 7 5 0 0) , and the er-zyine glutamate dehydrogeriase GDH (Xw 270000). The enzyme is inactive at 4OoC and fully activated at 3 0 O C. Independent of their particular primary structure ax! independent of the molecular weight (Mw) all three proteins investigated unfold at exactly the same temperature (113OC) The experimentally observed transition temperature Tm coincides with the extrapolated conversion temperature T* (Privalov, Privalov & Gill, Murphy and Gill, lreire and Murphy, 1-4) of 112OC. It is readily accepted that at this temperature the hydrophobic interactions will cease to contribute to the overall stability of the ordered secondary and/or tertiary structure causing the core of any globular protein to unfold to the denatured state. Thus 1 1 3 O C seems to be the threshold temperature for sustantation of life on earth.
Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temper... more Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122°C. The C-terminal 15-25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif (528 EKEKEKEGEK5 37) proved to be a key domain for stabilization at or near 100°C. Mutations ''tuned'' the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca 2+ , especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca 2+. The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.
The NADP +-specific glutamate dehydrogenase from Escherichia coli has been purified to electropho... more The NADP +-specific glutamate dehydrogenase from Escherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase from E. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The Km values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.
Page 1. Chapter 6 Key Enzymes in the Primary Nitrogen Metabolism of a Hyperthermophile Frank T. R... more Page 1. Chapter 6 Key Enzymes in the Primary Nitrogen Metabolism of a Hyperthermophile Frank T. Robb1, Yaeko Masuchi1, Jae-Bum Park2, and Michael WW Adams2 1Center for Marine Biotechnology, University of Maryland ...
Recombinant Protein Expression: Prokaryotic Hosts and Cell-Free Systems, 2021
Expression of heterologous genes in Escherichia coli is a routine technology for recombinant prot... more Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.
We describe circular polarization as a remote sensing diagnostic of chiral signatures which may b... more We describe circular polarization as a remote sensing diagnostic of chiral signatures which may be applied to Mars. The remarkable phenomenon of homochirality provides a unique biosignature which can be amenable to remote sensing through circular polarization spectroscopy. The natural tendency of microbes to congregate in close knit communities would be beneficial for such a survey. Observations of selected areas of the Mars surface could reveal chiral signatures and hence explore the possibility of extant or preserved biological material. We describe a new instrumental technique that may enable observations of this form.
The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic ar... more The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level ...
All of the important catalytic tasks in living cells are carried out by proteins folded into conf... more All of the important catalytic tasks in living cells are carried out by proteins folded into conformations that are essential for the biological function of the cell.
This report was prepared as an account of work sponsored by an agency of the United States Govern... more This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employes, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors exprcssed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
F1000 - Post-publication peer review of the biomedical literature, 2009
The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encode... more The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origens. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and-200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
F1000 - Post-publication peer review of the biomedical literature, 2008
We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pres... more We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO 2. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (؊34‰ to؊27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<؊12‰), and the kinetic isotope effect at 122°C and 40 MPa was ؊9.4‰, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane. carbon isotope fractionation ͉ deep-sea hydrothermal vent ͉ hyperthermophile ͉ methanogenesis ͉ piezophilic
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