Biology subjects

Melnik, B. S.

Publications and source records attributed to Melnik, B. S..

3 recordsLinked to original sources

bioRxiv2020

Designing and studying a mutant form of the ice-binding protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some organisms, helping them to survive extremely low temperatures. One of the problems in study of such proteins is the difficulty of isolation and purification. For example, eight cysteine residues in cfAFP from Choristoneura fumiferana (the eastern spruce budworm) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. In this work we designed a mutant form of ice-binding protein cfAFP, which is much more easy to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and at least does not decrease the freezing temperature.

molecular biology↗

bioRxiv2019

Loops linking secondary structure elements affect the stability of molten globule intermediate state of apomyoglobin

Apomyoglobin is a protein widely used as a model for studying globular protein folding. This work aimed to test the hypothesis on influence of rigidity and length of loops linking protein secondary structure elements on the stability of molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of proline residues to glycine and with loops elongated by three and six glycine residues. For all the protein forms, denaturation/renaturation kinetic curves at different urea concentrations were obtained, folding/unfolding constants were calculated and dependencies of rate constant logarithms on urea concentrations were plotted. All the data gave an opportunity to calculate free energies of different apomyoglobin states. As a result, the mutations in apomyoglobin loops were demonstrated to have a real effect on intermediate state stability compared to unfolded state.

biophysics↗

bioRxiv2019

GFP fusion protein with embedded foreign peptide

From the point of view structural biology and protein engineering the green fluorescent protein (GFP) is an exceptionally attracting object. The tertiary structure of GFP is quite unique: it reminds a "cylinder" or a "barrel" consisting of beta-layers that contains an alpha-helix inside. The "barrel" is a special container for an alpha-helix serving to protect the latter from the influence of the surroundings. Therefore a reasonable question arises whether the "barrel" can function as a container for preservation and isolation of other peptides. The alpha-helix itself contains hydrophilic amino acids, whereas inside the barrel there are many molecules of bound water. We supposed that the central alpha-helix of green fluorescent protein could be substituted for foreign peptide. In this study we checked the possibility for creation of such a system on base of GFP, where the toxic peptide is isolated from the environment inside the protein. The modification of green fluorescent protein was carried out. An antimicrobial peptide was inserted into the central alpha-helix. The results of our experiments show that such a chimeric protein is compact, soluble and non-toxic for the producing cell culture, but its structure is destabilized. The obtained data show that the idea of use of green fluorescent proteins as a < > for storing foreign peptides could be realized.

bioengineering↗