Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF
Abstract
:1. Introduction
2. Results
2.1. Analysis of Morphology of Aggregates
2.2. Small Angle X-Ray Scattering (SAXS) Data
2.3. Dimerization of BIF Molecules: Role of Surface Residue Substitutions
3. Discussion
4. Materials and Methods
4.1. Construction of the BIF-Expressing Plasmid pETBIF
4.2. Site-Directed Mutagenesis
4.3. Expression and Purification of the Recombinant BIF Protein and Its Mutant Analogues
4.4. Refolding of the Recombinant BIF Protein and Its Mutant Analogues
4.5. Formation of Aggregates of BIF Protein and Its Mutant Analogues in Solution
4.6. The Thioflavin T (ThT) Assay
4.7. Congo Red Spectroscopic Assay
4.8. Electrophoretic Analysis of Aggregates in Polyacrylamide Gel (PAGE) with 0.1% Sodium Dodecyl Sulfate
4.9. AFM Measurements
4.10. Small Angle X-Ray Scattering (SAXS)
4.11. The Congo Red Birefringence Assay
4.12. Molecular Modelling
4.13. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Protein | pH | tgα (SAXS), Type of Aggregates | Binding of ThT * (30 Days) | Binding of CR * (30 Days) | Resistance to 2% SDS (30 Days) | Birefringence of CR (30 Days) |
---|---|---|---|---|---|---|
BIF | 7.2 | −2.84 Mainly branched flat associates (14 days) −1.1 Mainly fibrillar structures (30 days) | + | + | + | + |
6.5 | “—” * | + | + | + | + | |
4.5 | −3.42 Mainly branched structures from globular particles | − | − | − | − | |
K170N | 7.2 | −2.0 Mainly branched flat associates (30 days) | − | − | − | − |
6.5 | −0.92 Rod-like structures (30 days) | − | − | − | − | |
4.5 | “—” | − | − | − | − | |
Y187N | 7.2 | −1.7 Mainly rod-like structures (30 days) | + | + | + | + |
6.5 | “—” | + | + | + | + | |
4.5 | “—” | + | + | + | + | |
R109N | 7.2 | “—” | + | + | + | + |
6.5 | “—” | + | + | + | + | |
4.5 | “—” | − | − | − | − | |
S157Q | 7.2 | −2.8 Mainly branched flat associates (14 days) | + | + | + | + |
6.5 | “—” | − | − | − | − | |
4.5 | “—” | − | − | − | − | |
D29Q | 7.2 | “—” | + | + | + | + |
6.5 | “—” | − | − | − | − | |
4.5 | “—” | − | − | − | − |
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Timchenko, M.; Abdullatypov, A.; Kihara, H.; Timchenko, A. Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF. Int. J. Mol. Sci. 2019, 20, 5197. https://doi.org/10.3390/ijms20205197
Timchenko M, Abdullatypov A, Kihara H, Timchenko A. Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF. International Journal of Molecular Sciences. 2019; 20(20):5197. https://doi.org/10.3390/ijms20205197
Chicago/Turabian StyleTimchenko, Maria, Azat Abdullatypov, Hiroshi Kihara, and Alexander Timchenko. 2019. "Effect of Single Amino Acid Substitutions by Asn and Gln on Aggregation Properties of Bence-Jones Protein BIF" International Journal of Molecular Sciences 20, no. 20: 5197. https://doi.org/10.3390/ijms20205197