Improving the cell-membrane-penetrating activity of globins by introducing positive charges on protein surface: A case study of sperm whale myoglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.

A facile gelator based on phenylalanine derivative is capable of forming fluorescent Zn-metallohydrogel, detecting Zn2+ in aqueous solutions and imaging Zn2+ in living cells.

Supramolecular hydrogels are attracting soft materials with potential applications. In this study, we synthesized a facile gelator (named 2-QF) based on phenylalanine derivative with a Quinoline group. 2-QF can assemble to form hydrogels at room temperature in different colors under low pH values. Moreover, 2-QF was triggered to form a yellow metallohydrogel (2-QF-Zn) at high pH by the coordination between 2-QF and Zn2+. 2-QF-Zn metallohydrogel showed excellent multi-stimuli responsiveness, especially the reversible "on-off" luminescence switching, as induced by base/acid. In addition, at a low concentration, 2-QF can selectively and visibly identify Zn2+ through fluorescence enhancement, and can detect Zn2+ at physiological pH as a chemosensor. Remarkably, 2-QF and 2-QF-Zn exhibited an excellent biocompatibility without cell cytotoxicity, and 2-QF is able to penetrate live HeLa cells and image intracellular Zn2+ by a turn-on fluorescent response, which makes it a potential candidate for biomedical applications.

Efficient biodegradation of malachite green by an artificial enzyme designed in myoglobin.

Synthetic dyes such as malachite green (MG) have a wide range of applications. Meanwhile, they bring great challenges for environmental security and cause potential damages to human health. Compared with traditional approaches, enzymatic catalysis is an emerging technique for wastewater treatment. As alternatives to natural enzymes, artificial enzymes have received much attention for potential applications. In previous studies, we have rationally designed artificial enzymes based on myoglobin (Mb), such as by introducing a distal histidine (F43H mutation) and creating a channel to the heme pocket (H64A mutation). We herein show that the artificial enzyme of F43H/H64A Mb can be successfully applied for efficient biodegradation of MG under weak acid conditions. The degradation efficiency is much higher than those of natural enzymes, such as dye-decolorizing peroxidase and laccase (13-18-fold). The interaction of MG and F43H/H64A Mb was investigated by using both experimental and molecular docking studies, and the biodegradation products of MG were also revealed by UPLC-ESI-MS analysis. Based on these results, we proposed a plausible biodegradation mechanism of MG. With the high-yield of overexpression in E. coli cells, this study suggests that the artificial enzyme has potential applications in the biodegradation of MG in fisheries and textile industries.