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The mutated recombinant kinase domain of human fibroblast growth factor receptor 2b (hFGFR2b) is overexpressed and purified, and its structural changes upon the interaction with three unsaturated fatty acids (UFAs), oleic, linoleic and α-linolenic are studied. This interaction is investigated to find out about the folding and unfolding effect of unsaturated fatty acids on the kinase domain structure of hFGFR2b. Recombinant pLEICS-01 vectors, containing the mutated coding region of hFGFR2b, are expressed in the standard Escherichia coli BL21 (DE3) host cells and purified by Ni-NTA affinity chromatography. While polyacrylamide gel electrophoresis characterizes the functionality of recombinant protein, its structural changes are studied in the presence and absence of various concentrations of oleic, α-linolenic and linoleic acids using circular dichroism (CD) and fluorescence spectroscopy. Far ultraviolet CD results show that unsaturated fatty acids do not change the secondary structure of the recombinant kinase domain of hFGFR2b. However, chemical denaturation analysis confirms that all three UFAs destabilize the tertiary structure of recombinant protein. A decrease in the fluorescence intensity without any significant red or blue shift (336±1nm) reflects a variation in the tertiary structure of protein. The direct interaction of the studied UFAs with hFGFR2b reduces the conformational stability of their kinase domains. The structural changes in hFGFR2b in the presence of UFAs may be necessary for hFGFR2b to adjust the signal transduction and regulate the key cellular processes.

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.

Tyrosinase is a determinant enzyme for modulating melanin production as its abnormal activity can result in an increased amount of melanin. Reduction of tyrosinase activity has been targeted for preventing and healing hyperpigmentation of skin, such as melanoma and age related spots. The aim of this systematic study is to investigate whether recombinant S100A8/A9 and its modified form reduce the activity of mushroom tyrosinase (MT) through changing its structure. Recombinant His-Tagged S100A8 and S100A9 are expressed in Escherichia coli BL21 (DE3) and modified using Woodward's reagent K which is a carboxyl group modifier. The structures of S100A8/A9 and its modified form are studied using fluorescence and circular dichroism spectroscopy, and the activity of MT is measured using UV-visible spectrophotometry in the presence of its substrate, L-3,4-dihydroxyphenylalanine (L-DOPA). The results show a lower stability of the modified protein when compared with its unmodified form. The interaction of S100A8/A9 with MT changes the structure and successfully reduces the activity of mushroom tyrosinase. Recombinant S100A8/A9 complex decreases MT activity which can control malignant melanoma, the most dangerous type of skin cancer.