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Despite research efforts to unravel the role of naturally occurring deep eutectic solvents (NADESs) in living cells, there are still many questions left unanswered. The proposed hypothesis about the role of NADESs in cryopreservation and resistance of cells motivated us to investigate whether DESs might possess chaperoning activity. Accordingly, here, the chaperone‐like activity of a well‐known DES, urea‐choline chloride DES (UC), on a model protein, lysozyme, has been evaluated. The results showed that UC DES was not only capable of suppressing lysozyme aggregation almost completely, but also efficiently enhanced the refolding yield of the denatured enzyme by nearly 95%, thereby suggesting the chaperoning potency of DESs. The refolding promotion of lysozyme was also verified through fluorescence study. The results may introduce DESs as new generation chaperones and shed light on their role in cell resistance under stress conditions.

Artemin is an abundant thermostable protein in Artemia encysted embryos under stress. It is considered as a stress protein, as its highly regulated expression is associated with stress resistance in this crustacea. In the present study, artemin has been shown to be a potent molecular chaperone with high efficacy. Artemin is capable of inhibiting the chemical aggregation of proteins such as carbonic anhydrase (CA) and horseradish peroxidase (HRP) at unique molar ratios of chaperone to substrates (1:40 and 1:26 for CA and HRP, respectively). Furthermore, it can also enhance refolding yield of these substrates by nearly 50%. The refolding promotion of CA is checked and verified through a sensitive fluorimetric technique. Based on these experiments, artemin showed higher chaperone activity than other chaperones. The evaluation of artemin surface using ANS showed it to be highly hydrophobic, probably resulting in its high efficacy. These results suggest that artemin can be considered a novel low molecular weight chaperone.

Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently‐crystallized VEGF/VEGF receptor‐2 (VEGFR‐2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR‐2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two‐step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng·mL−1, respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti‐angiogenic potency of this VEGF antagonist may allow its future use as an anti‐tumor agent. Structured digital abstract WT-VEGF and M-VEGF  bind by comigration in sds page ( View interaction).