Kinetics of Mutant Apomyoglobin Association

Abstract

Association of protein molecules in human tissues underlies many diseases, which brings it in the focus of numerous studies. Previous reports described amyloid formation with the unfolded protein state taken as the starting point. Here, we present kinetics of protein aggregation starting from the protein native state and with consideration of its structural stability. We constructed mutant forms of apomyoglobin (apoMb) and investigated them by light scattering, small-angle Xray scattering (SAXS), transmission electron microscopy (TEM), atomic force microscopy (AFM), fluorescence, far UV CD and FTIR spectroscopy. We found that apoMb mutants formed aggregates that changed their shape after 24 h incubation at a physiologically relevant temperature of 40 8C, pH 5.5, from almost globular (micellar) to elongated and curly. The elongated particles exhibited properties of the cross-beta structure characteristic of amyloids. According to SAXS, the elongated aggregates were not less than 300 Å in length, the gyration radius of their cross-section (R c ) was about 35 Å, and their molecular mass was over 400 kDa. An elongated shape of the aggregates was also demonstrated by TEM-and AFM imaging. The ability of apoMb mutants to form elongated amyloid-like aggregates, as well as their compactness, negatively correlated with stability of their native structure.

Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF

“…The tendency of proteins to aggregation complicated the performance of SAXS experiments; however, some information on their conformation in aggregates can be obtained, as was demonstrated by us for myoglobin [13]. We investigated conformation of oligomers plotting dependence logI-logQ where; I—intensity, Q—module of scattering vector.…”

sw ApoMb Amyloid Aggregation under Nondenaturing Conditions: The Role of Native Structure Stability

“…Previously, we showed that aggregation of WT sw ApoMb and its mutants is over in less than 24 h, and all recorded parameters (ThT fluorescence intensity, shape of FTIR spectra, and aggregate morphology) reach a plateau (37,64). However, during this time, some studied proteins do not completely convert to aggregates.…”