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As has been recently shown, the toxicity of protein aggregates is determined by their structure. Therefore, special attention has been focused on the search for factors that specify the structural features of formed amyloid fibrils. The effect of amino acid substitutions in apomyoglobin on the structural characteristics of its amyloid aggregates has been analyzed. The morphology and secondary structure of amyloids of the wild-type protein and its mutant variants Val10Ala, Val10Phe, and Trp14Phe have been compared, and the regions involved in intermolecular interactions in fibrils have been determined using limited proteolysis and mass spectrometry. No considerable differences have been found in the morphology (shape, length, or diameter) or the content (percentage) of the cross-β structure of apomyoglobin amyloids and its mutant variants. Amyloid cores of wild-type apomyoglobin and variants with Val10Phe and Trp14Phe substitutions have been formed by different regions of the polypeptide chain. The case study of apomyoglobin demonstrates that the location of amyloidogenic regions in the polypeptide chain of wild-type protein and its mutant forms can differ. Thus, possible structural changes in amyloids resulting from amino acid substitutions should be taken into account when studying phenotype aggregation.

The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and CD in the far UV-region. It is shown that a negatively charged phospholipid membrane can have a double effect on the structure of protein molecule upon their interaction: it denatures the native structure of the protein to its intermediate state similar to that in solution, acting as a moderately denaturing reagent. On the other hand, it can structure the unfolded protein to the same intermediate state stabilizing its structure. The kinetics of interaction between the protein and its mutant forms and the phospholipid membrane depends on the charge of the membrane surface. Here the rate of this interaction depends on the phospholipids vesicle concentration and the protein molecule stability increasing with a decrease of the latter. The importance of the obtained results for the folding of membrane proteins and the choice of the pathway for target delivery of protein drugs are discussed.

The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.

Three exon regions of the myoglobin gene of Baikal seal Phoca sibirica have been amplified and sequenced. The sequences have been found identical to those for the myoglobin gene of the North Atlantic seal Halichoerus grypus. Thus, these seal species have separated not earlier than 2.2 billion years before present.

The primary structure of myoglobin of the seal of Lake Baikal (East Siberia) Phoca siberica, determined by sequencing the whole protein and peptides obtained by the cyanogen bromide or proteinase cleavage and separated by the microcolumn liquid chromatography, was found to be identical to the primary structures of myoglobins of the harbour seal Phoca vitulina largha and the grey seal Halehoerus gryphus. It suggests that these species separated from a common ancestor less than seven million years ago.