Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8
Abstract
:1. Introduction
2. Results
2.1. Inclusion of Arg Residues in the Surface of the Constant Domain That Can Accommodate the Membrane Does Not Improve Antibody Potency
2.2. Requirements for Arg-Mediated 10E8 Potentiation and Complementation with HC-Ser100cPhe Mutation
2.3. LC Arg and HC S100cF Substitutions Promote Antibody–Membrane Interaction
2.4. 10E8 Substitutions Promote MPER-Binding in Model Membranes and Native Environments
3. Discussion
Concluding Remarks
4. Materials and Methods
4.1. Reagents
4.2. Antibody Production, Characterization and Labeling
4.3. Circular Dichroism
4.4. Enzyme-Linked Immunosorbent Assay (ELISA)
4.5. Pseudovirus Production
4.6. Cell-Entry Inhibition Assay
4.7. Transmission Infrared Spectroscopy
4.8. Single-Vesicle-Binding Assay
4.9. Isothermal Titration Calorimetry (ITC)
4.10. Surface Plasmon Resonance (SPR) Measurements
4.11. STED Imaging
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fab | KD (nM) | n | ΔH (kcal mol−1) | −TΔS (kcal mol−1) |
---|---|---|---|---|
WT | 32.39 | 1.12 | −6.73 | −3.49 |
2R | 46.29 | 1.17 | −6.24 | −3.75 |
2RF | 41.3 | 1.16 | −2.3 | −7.77 |
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Insausti, S.; Garcia-Porras, M.; Torralba, J.; Morillo, I.; Ramos-Caballero, A.; de la Arada, I.; Apellaniz, B.; Caaveiro, J.M.M.; Carravilla, P.; Eggeling, C.; et al. Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8. Int. J. Mol. Sci. 2022, 23, 10767. https://doi.org/10.3390/ijms231810767
Insausti S, Garcia-Porras M, Torralba J, Morillo I, Ramos-Caballero A, de la Arada I, Apellaniz B, Caaveiro JMM, Carravilla P, Eggeling C, et al. Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8. International Journal of Molecular Sciences. 2022; 23(18):10767. https://doi.org/10.3390/ijms231810767
Chicago/Turabian StyleInsausti, Sara, Miguel Garcia-Porras, Johana Torralba, Izaskun Morillo, Ander Ramos-Caballero, Igor de la Arada, Beatriz Apellaniz, Jose M. M. Caaveiro, Pablo Carravilla, Christian Eggeling, and et al. 2022. "Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8" International Journal of Molecular Sciences 23, no. 18: 10767. https://doi.org/10.3390/ijms231810767