Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Mutual effects of protein corona formation on CdTe quantum dots.

Future biomedical applications of nanoparticles will encounter these particles with patients' serum which might affect the properties and stability of quantum dots and serum proteins at the desired site of action. Therefore, it is essential to clarify the patient-specific serum components, serve as major interaction partners, the spatial distribution of these, and consequently the time-dependent effects of nanoparticle-protein interaction. Here, a biochemical and structural study was performed on the protein corona formation and the corresponding interaction of different sizes of CdTe QDs with human serum proteins to determine if the mutual effects on optical properties by using electrophoresis, chemiluminescence, and fluorescence spectroscopy. The results revealed that interaction with human serum significantly enhanced the stability and photoluminescence of quantum dots. Structural studies of HSA-coated CdTe QDs also showed that corona formation has no adverse effects on protein structure, and the reduction in fluorescence emissions of HSA is due to the direct quenching of aromatics residues by the quantum dot. Improving nanoparticle properties, as well as the lack of structural changes in HSA, can be very useful in biomedical applications and in vivo studies where stability is important.

Probing heat and oxidation induced conformational changes of molecular chaperone artemin by excitation-emission fluorescence spectroscopy.

Artemin is a potent molecular chaperone, which protects Artemia embryos undergoing encystment against extreme environmental stresses. In the present work, we have examined the structural changes of artemin from A. urmiana upon exposure to oxidant and heat, by using CD measurements as well as excitation-emission fluorescence spectroscopy as a powerful tool for monitoring the conformational transitions and molecular interactions in proteins. We have also provided here the first document on reporting the three dimensional fluorescence spectra of a protein using ANS. Totally, the fluorescence results indicated that the microenvironments of tyrosine and tryptophan residues and the hydrophobic pockets as well as the polypeptide backbone or secondary structure of the chaperone were influenced in responses to heat and H2O2 in different degrees. Moreover, the native state of artemin did not induce a considerable exposure of the internal non-polar groups to the solvent. Besides, the excitation-emission spectra of heated artemin by ANS revealed new emission peaks at 430-450 nm when it was excited at 330 nm, which suggests probable exposure of new binding sites for hydrophobic or electrostatic interactions of the protein with ANS. The protein also showed a greater conformational sensitivity to the temperature fluctuations compared to oxidation. Here, we presented some evidence in support of the relation between artemin and its stress dependent activation in vitro and in vivo. This study can expect that the EEM fluorescence spectroscopy could provide a promising tool to study conformational transitions of proteins.

PCR-based gene synthesis, molecular cloning, high level expression, purification, and characterization of novel antimicrobial peptide, brevinin-2R, in Escherichia coli.

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.

Structural studies of hen egg-white lysozyme dimer: comparison with monomer.

A facile method for the formation of covalent bonds between protein molecules is zero length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, we report a cross-linking method for lysozyme and some structural studies as well as catalytic activity assay was performed on lysozyme dimer. The results showed that catalytic activity of lysozyme dimer was the same as monomer. Also, the GdnCl-induced equilibrium unfolding of hen egg-white lysozyme monomer and dimer at pH 2 was studied over a temperature range of 290.7-303.2 K by means of CD spectroscopy. The lack of coincidence between two unfolding curves at 222 and 289 nm in lysozyme dimer was observed, which suggested the existence of intermediate state in unfolding process, while lysozyme monomer showed a single cooperative transition. Thus, the thermodynamic parameters were estimated on the basis of two-state mechanism for lysozyme monomer and three-state one for lysozyme dimer. These results indicated that zero length cross-linking can stabilize the intermediate, so the population of intermediate increased. Our results offer a special opportunity to study the role of intermediates in protein folding mechanisms. In addition thermal unfolding of monomer and dimer in 222 nm was achieved.

The effect of vitamin e on splenocytes apoptosis of gamma- irradiated BALB/c mice.

Apoptosis, a physiologic mechanism to eliminate unwanted cells, is also induced by ionizing irradiation, through production of free radicals. It has been demonstrated that antioxidants such as vitamin E are able to protect cells from damage caused by free radicals. Taken together we found it reasonable to make an attempt to evaluate the protective effect of vitamin E against apoptosis. The irradiated mice received 1 Gy/day gamma radiation for one day (low dose) or for three successive days (high dose, 3Gy). The splenocytes were then isolated at 6, 14 and 24 h after exposure. DNA gel electrophoresis and DNA fragmentation assay were done in addition to the evaluation of splenocytes cytology.Our results showed that Vitamin E can reduce apoptosis against low dose irradiation. However it is not able to completely block programmed cell death in high dose irradiation.