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. 2023 Nov 23;28(23):7736.
doi: 10.3390/molecules28237736.

Influence of Amino Acid Substitutions in ApoMb on Different Stages of Unfolding of Amyloids

Natalya Katina  1   2 , Victor Marchenkov  1 , Natalya Ryabova  1 , Nelly Ilyina  1 , Natalia Marchenko  1 , Vitalii Balobanov  1 , Alexey Finkelstein  1
Affiliations

Influence of Amino Acid Substitutions in ApoMb on Different Stages of Unfolding of Amyloids

Natalya Katina et al. Molecules. .

Abstract

To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids' secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids' stability.

Keywords: amyloid stability; apomyoglobin; hydrophobicity; native electrophoresis; unfolding transition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Electron image of aggregates formed by V10F ApoMb (a); infrared spectra of aggregates and monomeric protein (b).
Figure 2
Figure 2
Far-UV CD spectra of the monomer and amyloid of ApoMb V10F at various urea concentrations (a–d), and normalized unfolding transitions of the monomer (e) and amyloid (f). Solid lines (e,f) are the result of a sigmoidal approximation of the experimental data.
Figure 3
Figure 3
Unfolding of amyloid secondary structure for ApoMb and its mutant variants.
Figure 4
Figure 4
Dependence of midpoints of CD denaturation transitions of ApoMb amyloids ([urea]1/2β-U) on the fractions of the native state (fN) under conditions appropriate for aggregation (a), on the changes in hydrophobicity as a result of mutation (b), and on the position of amino acid substitution (c).
Figure 5
Figure 5
BN-PAGE of ApoMb V10F amyloid solutions after their incubation at various concentrations of urea (a); total intensity of the lanes (b); fractions of amyloid (fAm), intermediate aggregates (fAgr), and monomeric protein (fM), as a function of urea concentrations (c), fraction of unfolded protein (fU), as a function of urea concentrations (d), supposed model of amyloids’ unfolding, where Amβ—amyloid with cross-β-structure, AmI—intermediate aggregates of molecular weight higher 1 MDa and without cross-β-structure, Agr—intermediate aggregate of 1 MDa, MU—unfolded monomer (e).
Figure 6
Figure 6
Dependence of the fractions of amyloids (a), aggregates (b) and monomeric proteins (c) for ApoMb and its mutant variants on urea concentration; molecular weights (MW) for protein markers and the BN-PAGE lanes of the V10F and L115F ApoMb variants’ amyloid in 8 M urea (d).
Figure 7
Figure 7
Dependence of midpoints of the amyloids’ (a) and intermediate aggregates’ (b) dissociation on the positions of the amino acid substitutions.
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