Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II
Abstract
:1. Introduction
1.1. How Mutations of a Protein Affect Its Ability to Form Amyloids?
1.2. What Parametric Plots Are and How to Interpret Them
2. Results and Discussion
2.1. Comparison of Unfolding Pathways of Mutant Forms of Carbonic Anhydrase II Using Time-Resolved Fluorescence Technique
2.2. Growth of Amyloid Structures of the Mutant Forms of Carbonic Anhydrase II
2.3. The Relationship between Mutation Effects on Unfolding Pathway of Carbonic Anhydrase and Its Amyloid Formation
- –
- Substitution L78A (in β-turn) slightly destabilizes both protein intermediates (Figure 3C,D) but has no effect on the structure of them (Figure 4A); this mutant form exhibits two-times-longer lag time (Figure 5C) and a slightly lower rate of amyloid growth kinetics (Figure 5D) as compared with wild-type protein;
- –
- Substitution L139A (in β-sheet) significantly decreases the intermediates’ stability (Figure 3C,D) without changing their structure (Figure 4A); for this mutant variant, a pronounced increase in the lag time (3.6-fold, Figure 5C) and a small decrease in the growth rate (Figure 5D) were found as compared with wild-type protein;
- –
- Substitution I208A (in β-turn), in addition to the destabilization of unfolding intermediates of carbonic anhydrase (Figure 3C,D), was found to disturb the structure of the late (molten globule-like, I2)-intermediate (Figure 4B); this mutant form demonstrates the fastest amyloid growth kinetics among all studied proteins (Figure 5C,D);
- –
- Substitution M239A (in unstructured C-terminal part) causes the lower stability of the protein-unfolding intermediates (Figure 3C,D) and changes the structure of the early-intermediate (I1) of carbonic anhydrase (Figure 4B); this mutant variant has a slower amyloids growth kinetics with about a two-times-longer lag time and two-times-lower growth rate (Figure 5C,D) than wild-type protein.
3. Materials and Methods
3.1. Isolation and Purification of Carbonic Anhydrase II and Its Mutant Forms
3.2. Preparation of Carbonic Anhydrase II Amyloids
3.3. SDS–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.4. Electron Microscopy
3.5. Fluorescence Thioflavin T Measurements
3.6. Steady-State and Time-Resolved Fluorescence Measurements
4. Conclusions and Proposals
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Melnik, B.S.; Katina, N.S.; Ryabova, N.A.; Marchenkov, V.V.; Melnik, T.N.; Karuzina, N.E.; Nemtseva, E.V. Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II. Int. J. Mol. Sci. 2022, 23, 14645. https://doi.org/10.3390/ijms232314645
Melnik BS, Katina NS, Ryabova NA, Marchenkov VV, Melnik TN, Karuzina NE, Nemtseva EV. Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II. International Journal of Molecular Sciences. 2022; 23(23):14645. https://doi.org/10.3390/ijms232314645
Chicago/Turabian StyleMelnik, Bogdan S., Natalya S. Katina, Natalya A. Ryabova, Victor V. Marchenkov, Tatiana N. Melnik, Natalya E. Karuzina, and Elena V. Nemtseva. 2022. "Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II" International Journal of Molecular Sciences 23, no. 23: 14645. https://doi.org/10.3390/ijms232314645