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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.

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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.

1Artemin is an abundant thermostable protein in Artemia embryos and considered as a highly 2 efficient molecular chaperone against extreme environmental stress conditions. The dynamic 3 conformational properties of artemin appear to play a critical role in its biological activities. In 4 this study, we have investigated the conformational transitions and functional changes of artemin 5 under heat and oxidative stress to find some evidence of the relationship between the structure 6 and function of artemin. The tertiary and quaternary structures of artemin have been evaluated by 7 fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based 8 on the structural analysis, artemin showed irreversible substantial conformational lability in 9 response to heat and oxidant which mainly mediated through the hydrophobic interactions and 10 dimerization of the chaperone. In addition, the chaperone-like activity of the heated and oxidized 11 artemin was examined using lysozyme refolding assay and the experiments showed that although 12 both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous 13 incubation with both stressors significantly triggered the activation of artemin. Moreover, the 14 heat-induced dimerization of artemin was found to be the most critical factor for its activation. It 15 was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin 16 through formation of disulfide bridges between the subunits and strengthens its chaperoning 17 efficacy. Accordingly, it was proposed that artemin probably exists in a monomer-oligomer 18 equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein 19 substrates may shift the equilibrium towards the active dimer forms of the chaperone. 20 Keywords: Artemin, heat and oxidative stress, conformational transitions.21 22 STATEMENT OF SIGNIFICANCE 1There are a number of reports in which the chaperone-like activity of artemin, as a stress protein, 2 has been confirmed in vivo and in vitro. Nonetheless, the details of structural changes of the 3 protein have not been fully discovered yet. In the present work, we focused on conformational 4 properties of artemin from A. urmiana upon exposing to heat and oxidation, by using various 5 structural and functional analysis in order to predict the mechanisms of artemin's activation. 6 Notably, this is the first document on reporting the structural transitions of the chaperone in 7 stress conditions. 8 9 1Artemin is a stress protein of encysted Artemia embryos, representing about 10% of the soluble 2 cellular proteins, but it is almost completely absent from nauplius larvae (1). Due to high 3 structural stability, abundance and chaperone function, artemin probably contributes to cyst 4 stress resistance (2). Artemin monomers consist of 229 amino acid residues and exhibit a 5 molecular mass of 26 kDa and assemble into rosette-like oligomers of ~600-700 kDa consist of 6 24 monomer subunits (2,3). Arte...