Abstract
NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer–dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.
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Data availability
All AUC data (primary sedimentation velocity data, fitted model results, and reports) are stored in the UltraScan LIMS database at the Canadian Center for Hydrodynamics.
Change history
03 April 2023
In Abstract, the sentence “The biophysical properties of NET-1+UNC-5B complex has been poorly characterized” was incorrect. Now, it has been corrected to “The biophysical properties of the NET-1+UNC-5B complex have been poorly characterized”.
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Acknowledgements
This work was supported by the Canada 150 Research Chairs program (C150-2017-00015), the Canada Foundation for Innovation (CFI-37589), the National Institutes of Health (1R01GM120600) and the Canadian Natural Science and Engineering Research Council (DG-RGPIN-2019-05637). UltraScan supercomputer calculations were supported through NSF/XSEDE grant TG-MCB070039N, and University of Texas grant TG457201. (All grants to B.D.). JS is a Tier-1 Canada Research Chair in Structural Biology and Biophysics. This research is funded by the Canadian Institutes of Health Research (CIHR)—Grant 201610PJT-152935 (JS).
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HG performed AUC experiments, and HG and BD analyzed the data. MM assisted in AUC data acquisition. FH performed MD simulations. MG and MK recombinantly expressed, and purified all protein samples. JS and BD supervised the project. HG, JS and BD wrote the manuscript.
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Special Issue: Analytical Ultracentrifugation 2022.
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249_2023_1644_MOESM1_ESM.tif
Supplementary file1 (TIF 379 KB) The differential scanning fluorimetry results showing the melting curve of NET-1 in the presence of 1 mM CaCl2 (red) and 1 mM EDTA (blue). The melting temperature of NET-1 in the presence of 1 mM EDTA is 38.8 °C (vertical blue line) and in the presence of 1 mM CaCl2 the melting temperature is 50.4 °C (vertical red line)
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Gabir, H., Gupta, M., Meier, M. et al. Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation. Eur Biophys J 52, 473–481 (2023). https://doi.org/10.1007/s00249-023-01644-1
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DOI: https://doi.org/10.1007/s00249-023-01644-1