RESUMO
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-ß 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.
Assuntos
Amiloide/química , Apoproteínas/química , Mioglobina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Mioglobina/genética , Estrutura Secundária de ProteínaRESUMO
A method for the synthesis and high purification of fragments of Aß(1-42) peptide has been elaborated. We have synthesized the amyloidogenic fragment Aß(16-25) predicted by us and studied the process of its aggregation by electron microscopy and X-ray analysis. Electron microscopy images show that the peptide forms a film, which is not characteristic of amyloid fibrils. At the same time, according to the X-ray diffraction data, its preparations display the presence of two main reflections (4.6-4.8 and 8-12 Å) characteristic of cross-ß structure of amyloid fibrils. Thus, the fragment Aß(16-25) that we predicted is a promising object not only for studying the process of polymerization of the peptides/proteins, but also for using it as a nanomaterial to study a number of biological processes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Nanoestruturas/química , Amiloide/química , Amiloide/metabolismo , Cristalografia por Raios X , Microscopia Eletrônica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aß(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aß(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/química , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Microscopia Eletrônica , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Conformação Proteica , Proteólise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We have developed a highly efficient method for purification of the recombinant product Aß(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aß(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one "monomer" and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.
