Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning and Site-Directed Mutagenesis
2.2. Protein Expression and Purification
2.3. Enzymatic Activity Assays
2.4. Circular Dichroism (CD) Thermal Denaturation Analysis
2.5. Protein Crystallization
2.6. Data Collection, Structure Solution and Refinement
2.7. Protein Data Bank (PDB) Deposition
3. Results
3.1. Variant Production
3.2. Kinetic Characterization
3.3. Circular Dichroism (CD) Thermal Denaturation Analysis
3.4. Structural Characterization of the HT-hTS Variant Q62R
3.4.1. The HT-hTS Q62R Active Site
3.4.2. The Arg62 Pocket
4. Discussion
4.1. 5-FTHF Binding in the HT-hTS Q62R Active Site
4.2. Effect of the Interface Point Mutation Q62R on the hTS Dimer Stability
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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KM (dUMP) (μM) | KM (mTHF) (μM) | kcat (s−1) | kcat/KM (dUMP) (μM−1 s−1) | kcat/KM (mTHF) (μM−1 s−1) | |
---|---|---|---|---|---|
HT-hTS | 10 ± 1 | 6 ± 1 | 1.00 ± 0.01 | 1.00 × 10−7 | 1.67 × 10−7 |
HT-hTS Q62R | 8 ± 1 | 16 ± 1 | 0.16 ± 0.06 | 0.20 × 10−7 | 0.10 × 10−7 |
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Pozzi, C.; Lopresti, L.; Santucci, M.; Costi, M.P.; Mangani, S. Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R. Biomolecules 2019, 9, 134. https://doi.org/10.3390/biom9040134
Pozzi C, Lopresti L, Santucci M, Costi MP, Mangani S. Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R. Biomolecules. 2019; 9(4):134. https://doi.org/10.3390/biom9040134
Chicago/Turabian StylePozzi, Cecilia, Ludovica Lopresti, Matteo Santucci, Maria Paola Costi, and Stefano Mangani. 2019. "Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R" Biomolecules 9, no. 4: 134. https://doi.org/10.3390/biom9040134
APA StylePozzi, C., Lopresti, L., Santucci, M., Costi, M. P., & Mangani, S. (2019). Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R. Biomolecules, 9(4), 134. https://doi.org/10.3390/biom9040134