Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation

Haben Gabir; Monika Gupta; Markus Meier; Fabian Heide; Manuel Koch; Joerg Stetefeld; Borries Demeler

doi:10.1007/s00249-023-01644-1

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation

Haben Gabir
, Monika Gupta
, Markus Meier
, Fabian Heide
, Manuel Koch
, Joerg Stetefeld
, Borries Demeler

Chemistry and Biochemistry

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

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.

Original language	English
Pages (from-to)	473-481
Number of pages	9
Journal	European Biophysics Journal
Volume	52
Issue number	4-5
DOIs	https://doi.org/10.1007/s00249-023-01644-1
State	Published - Jul 2023

Funding

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).

Funders	Funder number
TG457201
TG-MCB070039N
1R01GM120600
Canadian Institutes of Health Research	201610PJT-152935
DG-RGPIN-2019-05637
Canada Foundation for Innovation	CFI-37589
C150-2017-00015

Keywords

Axon guidance
Hetero-interaction studies between proteins
Hydrodynamic bead modeling
Multi-wavelength analytical ultracentrifugation
Netrin-1
UltraScan
UltraScan-SOMO
Uncoordinated-5

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10.1007/s00249-023-01644-1

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title = "Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation",

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.",

keywords = "Axon guidance, Hetero-interaction studies between proteins, Hydrodynamic bead modeling, Multi-wavelength analytical ultracentrifugation, Netrin-1, UltraScan, UltraScan-SOMO, Uncoordinated-5",

author = "Haben Gabir and Monika Gupta and Markus Meier and Fabian Heide and Manuel Koch and Joerg Stetefeld and Borries Demeler",

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year = "2023",

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doi = "10.1007/s00249-023-01644-1",

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T1 - Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation

AU - Gabir, Haben

AU - Gupta, Monika

AU - Meier, Markus

AU - Heide, Fabian

AU - Koch, Manuel

AU - Stetefeld, Joerg

AU - Demeler, Borries

PY - 2023/7

Y1 - 2023/7

N2 - 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.

AB - 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.

KW - Axon guidance

KW - Hetero-interaction studies between proteins

KW - Hydrodynamic bead modeling

KW - Multi-wavelength analytical ultracentrifugation

KW - Netrin-1

KW - UltraScan

KW - UltraScan-SOMO

KW - Uncoordinated-5

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DO - 10.1007/s00249-023-01644-1

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SN - 0175-7571

VL - 52

SP - 473

EP - 481

JO - European Biophysics Journal

JF - European Biophysics Journal

IS - 4-5

ER -

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation

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