ABSTRACT
The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.
Subject(s)
Copper/analysis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Histidine/chemistry , Spectrometry, Fluorescence/methods , Fluorescence , Hydrogen-Ion Concentration , Models, Molecular , ProtonsABSTRACT
The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.
Subject(s)
Protein Folding , Protein Multimerization , Tumor Necrosis Factors/metabolism , Brain/cytology , Brain/metabolism , DNA, Complementary/genetics , DNA, Complementary/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Glucocorticoid-Induced TNFR-Related Protein/metabolism , Humans , Inclusion Bodies/metabolism , Ligands , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Necrosis Factors/genetics , U937 CellsABSTRACT
Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.
Subject(s)
Copper/chemistry , Green Fluorescent Proteins/chemistry , Histidine/chemistry , Spectrometry, Fluorescence/methods , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins/genetics , Hydrogen-Ion Concentration , Models, Molecular , Mutagenesis, Site-Directed , Plasmids , TemperatureABSTRACT
Human serum transferrin has a potential for drug-delivery systems. Oxalate and aziridine-carboxylate was conjugated to serum transferrin in order to transport into the targeted cancer cells via transferrin-receptor mediated endocytosis. Capillary zone electrophoresis and capillary isoelectric focusing were used to analyze the effectiveness of complexation reactions. The electropherograms show the differences between iron-free- and iron-complexed molecular forms of human serum transferrin. The iron-complexed transferrin sample containing the different anions as synergistic complexing agents were characterized by different electrophoretic parameters.
Subject(s)
Anions/chemistry , Antineoplastic Agents/pharmacology , Iron/chemistry , Transferrin/chemistry , Animals , Anions/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Aziridines/chemistry , Aziridines/metabolism , Aziridines/pharmacology , Biological Transport , Cell Line, Tumor , Drug Delivery Systems , Electrophoresis, Capillary , Humans , Iron/metabolism , Isoelectric Focusing , Oxalates/chemistry , Oxalates/metabolism , Oxalates/pharmacology , Protein Binding , Transferrin/metabolismABSTRACT
Supercritical carbon dioxide (CO(2)) possesses germicide (bactericide and sporicide) effect. Despite of the fact, that this effect is used in industrial sterilization processes, the sterilization mechanism at molecular level is unclear. Our hypotheses can provide a molecular-biological explanation for the phenomenon. We believe that in supercritical state CO(2) reacts competitively with Met-tRNA(fMet), the formation rate and the amount of formyl-methionyl-tRNA (fMet-tRNA(fMet)) will be diminished by irreversible substrate consumption. The fMet-tRNA(fMet) possesses a key role in prokaryotic protein synthesis, being almost exclusively the initiator aminoacyl-tRNA. The formed carbamoyl-methionyl-tRNA (cMet-tRNA(fMet)), probably stable only under pressure and high CO(2) concentration, is stabilized by forming a ternary molecular complex with the GTP-form of the translational initiation factor 2 (GTP-IF2). This complex is unable to dissociate from preinitiation 70S ribosomal complex because of strong polar binding between the protein C-2 domain and the modified initiator aminoacyl-tRNA. The IF2-fMet-tRNA(fMet)-blocked 70S ribosomal preinitiation complex does not decompose following the GTP hydrolysis, becoming unable to synthesize proteins. The death of the microbial cell is caused by inhibition of the protein synthesis and energetic depletion. Moreover, we propose a possible mechanism for the accumulation of cMet-tRNA(fMet) in the bacterial cell. Since the translational process is an important target for antibiotics, the proposed mechanism could be a work hypothesis for discovery of new antibiotics. Made by high conservative character of prokaryotic translation initiation, the proposed IF2 pathway deterioration strategy may conduct to obtaining selective (with low mammalian toxicity) antimicrobials and at the same time, with reduced possibility of the drug resistance development.
