Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.

ABSTRACT: The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4°C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV-visible absorption spectroscopy. The melting temperature (Tm) and the van't Hoff enthalpy (?HvH) were determined. These values suggest an unexpectedly high stability for this psychrophilic cytochrome.

SUBMITTER: Oswald VF

PROVIDER: S-EPMC3885257 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.

Oswald Victoria F VF Chen WeiTing W Harvilla Paul B PB Magyar John S JS

Journal of inorganic biochemistry 20131112

The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4°C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV-visible absorption spectroscopy. The melting temperature (Tm) ...[more]

PMID: 24275750

Similar Datasets

The structure of ferricytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H.

Project description:Approximately 40% of all proteins are metalloproteins, and approximately 80% of Earth's ecosystems are at temperatures ≤5 °C, including 90% of the global ocean. Thus, an essential aspect of marine metallobiochemistry is an understanding of the structure, dynamics, and mechanisms of cold adaptation of metalloproteins from marine microorganisms. Here, the molecular structure of the electron-transfer protein cytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H has been determined by X-ray crystallography (PDB: ). The structure is highly superimposable with that of the homologous cytochrome from the mesophile Marinobacter hydrocarbonoclasticus. Based on structural analysis and comparison of psychrophilic, psychrotolerant, and mesophilic sequences, a methionine-based ligand-substitution mechanism for psychrophilic protein stabilization is proposed.

| S-EPMC4243462 | biostudies-literature

Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

Project description:We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

| S-EPMC5339861 | biostudies-literature

Identification and molecular characterization of a psychrophilic GH1 β-glucosidase from the subtropical soil microorganism Exiguobacterium sp. GXG2.

Project description:The products of bacterial β-glucosidases with favorable cold-adapted properties have industrial applications. A psychrophilic β-glucosidase gene named bglG from subtropical soil microorganism Exiguobacterium sp. GXG2 was isolated and characterized by function-based screening strategy. Results of multiple alignments showed that the derived protein BglG shared 45.7% identities with reviewed β-glucosidases in the UniProtKB/Swiss-Prot database. Functional characterization of the β-glucosidase BglG indicated that BglG was a 468 aa protein with a molecular weight of 53.2 kDa. The BglG showed the highest activity in pH 7.0 at 35 °C and exhibited consistently high levels of activity within low temperatures ranging from 5 to 35 °C. The BglG appeared to be a psychrophilic enzyme. The values of Km, Vmax, kcat, and kcat/Km of recombinant BglG toward ρNPG were 1.1 mM, 1.4 µg/mL/min, 12.7 s-1, and 11.5 mM/s, respectively. The specific enzyme activity of BglG was 12.14 U/mg. The metal ion of Ca2+ and Fe3+ could stimulate the activity of BglG, whereas Mn2+ inhibited the activity. The cold-adapted β-glucosidase BglG displayed remarkable biochemical properties, making it a potential candidate for future industrial applications.

| S-EPMC6773797 | biostudies-literature

Corrigendum: Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

Project description: Not available

| S-EPMC5422921 | biostudies-literature

Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain.

Project description:An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg(2+), Mn(2+), K(+); Ca(2+), Na(+), Ba(2+), Zn(2+), Cu(2+), Co(2+), Fe(2+), Sr(2+) and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

| S-EPMC4568857 | biostudies-literature

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

Project description:As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.

| S-EPMC1164895 | biostudies-other

Heme axial methionine fluxionality in Hydrogenobacter thermophilus cytochrome c552.

Project description:The heme group in paramagnetic (S = 1/2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole beta substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c(552) from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the beta substituents on all four heme pyrroles. Here, determination of the orientation of the magnetic axes and analysis of NMR line shapes for H. thermophilus ferricytochrome c(552) is performed. These data reveal that the unusual electronic structure for this protein is a result of fluxionality of the heme axial methionine. It is proposed that the ligand undergoes inversion at the pyramidal sulfur, and the rapid interconversion between two diastereomeric forms results in the unusual heme electronic structure. Thus a fluxional process for a metal-bound amino acid side chain has now been identified.

| S-EPMC423247 | biostudies-literature

Enthalpy arrays.

Project description:We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.

| S-EPMC470707 | biostudies-other