Mechanically transduced immunosorbent assay to measure protein-protein interactions
- Department of Biochemistry and Biophysics, The University of North Carolina School of Medicine, United States
- UNC Lineberger Comprehensive Cancer Center, University of North Carolina, United States
- Department of Chemistry, University of the Pacific, United States
- Department of Biological Engineering, University of the Pacific, United States
- Department of Mechanical Engineering, University of the Pacific, United States
Figures
Figure 1 with 3 supplements
Experimental schematic of the Mechanically Transduced Immunosorbent Assay (METRIS) used to measure protein-protein interactions.
(A) General schematic of roller and surface functionalization. Both binder A and binder B are attached to the roller or the surface by biotin-streptavidin interactions. The direction of the rotating …
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Figure 1—source data 1
Rolling parameter from all rolls for either biotin-streptavidin and avidin-streptavidin.
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
METRIS apparatus.
(A) Three pairs of Helmholtz coils were mounted on an aluminum T-slot assembly. Two sinusoidal signals are generated in Matlab, passed through a DAQ, amplifier, and then to the Helmholtz coils. …
Figure 1—video 1
Experimental video of streptavidin-coated rollers on an avidin-coated substrate and sped up 8X.
Figure 1—video 2
Experimental video of streptavidin-coated rollers on a biotin-coated substrate sped up 8X.
Figure 2 with 4 supplements
DIDO1-PHD interactions with H3 peptides characterized using METRIS.
(A) Plot showing the average translational displacement per actuation for the rollers coated with the indicated H3K4 methylated peptide on a DIDO1-PHD surface. Streptavidin-biotin and …
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Figure 2—source data 1
Rolling parameter from all rolls for the indicated rollers on a DIDO1-PHD surface. Each row is a different roller and each column is an actuation.
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig2-data1-v1.xlsx
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Figure 2—source data 2
Statistical analysis of results from METRIS measurements and histone peptide microarray results for DIDO1-PHD.
Results from statistical tests comparing the indicated pairs of rolling parameters (METRIS) or microarray results (Array).
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Rolling parameter for all rolls on the DIDO-PHD surface.
(A) Histogram showing the distribution of RPs for the indicated beads on the DIDO1-PHD-coated surface. See Figure 2—source data 1 for the rolling parameter for each actuation. (B) Normalized array …
Figure 2—video 1
Experimental video of H3.
Coated rollers on a DIDO1 PHD-coated substrate sped up 8X.
Figure 2—video 2
Experimental video of H3K4me3-coated rollers on a DIDO1 PHD-coated substrate sped up 8X.
Figure 2—animation 1
Animation of 30 rolls for each roller for all DIDO1-PHD family of interactions: Orange, H3K9me3; Purple, H3K9me2; Cyan, H3K9me1; Green, H3, Grey; H3T3pK4me3.
Streptavidin-avidin (red) and biotin-avidin (black) are included for reference. The video is then reversed so it can be looped.
Figure 3 with 4 supplements
ORC1-BAH domain interactions characterized using METRIS.
(A) Plot showing the average translational displacement per actuation for the rollers immobilized with the H4K20 methylated peptide on a ORC1-BAH domain surface. Streptavidin-Biotin and …
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Figure 3—source data 1
Rolling parameter from all rolls for the indicated rollers on a ORC1-BAH surface. Each row is a different roller and each column is an actuation.
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig3-data1-v1.xlsx
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Figure 3—source data 2
Statistical analysis of results from METRIS measurements and histone peptide microarray results for ORC1-BAH.
Results from statistical tests comparing the indicated pairs of rolling parameters (METRIS) or microarray results (Array).
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Rolling parameter for all rolls on the ORC1-BAH surface.
(A) Histogram showing the distribution of RPs for the indicated beads on the ORC1-BAH-coated surface. See Figure 3—source data 1 for the rolling parameter for each actuation. (B) Normalized array …
Figure 3—video 1
Experimental video of H4-coated rollers on a ORC1-BAH-coated substrate sped up 8X.
Figure 3—video 2
Experimental video of H4K20me2-coated rollers on a ORC1-BAH-coated substrate sped up 8X.
Figure 3—animation 1
Animation of 30 rolls for each roller for all ORC1-BAH family of interactions: Orange, H4K20me3; Purple, H4K20me2; Cyan, H4K20me1; Green, H4.
The video is then reversed so it can be looped.
Figure 4 with 2 supplements
Measuring the interaction of UBL domains with Ube2D1 or the UHRF1-SRA domain using METRIS.
(A) Plot showing the average translational displacement per actuation for the rollers coated with the indicated proteins on a surface containing UbeD21 (E2) or the UHRF1-SRA domain. …
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Figure 4—source data 1
Rolling parameter from all rolls for the indicated rollers on either a Ube2D1 surface or a UHRF1-SRA surface.
Each row is a different roller and each column is an actuation.
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig4-data1-v1.xlsx
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Figure 4—source data 2
Statistical analysis of results from METRIS measurements for ubiquitin and UHRF1-UBL binding to Ube2D1 or UHRF1-SRA domain.
Results from statistical tests comparing the indicated pairs of rolling parameters (METRIS).
- https://cdn.elifesciences.org/articles/67525/elife-67525-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Rolling parameter for all rolls on the Ube2D1 or SRA surface.
(A) Histogram showing the distribution of RPs for the indicated beads on the Ube2D1-coated surface. (B) Histogram showing the distribution of RPs for the indicated beads on the UHRF1-SRA-coated …
Figure 4—animation 1
Animation of 30 rolls for each roller for all Ube2d1 and UHRF1 UBL family of interactions: Cyan, UHRF1-UBL; Green, ubiquitin; both on a Ube2D1 surface.
Purple, UHRF1-UBL, Grey UHRF1-UBL W2V; Orange; Ubiquitin, all on the UHRF1-SRA surface. The video is then reversed so it can be looped.
Figure 5
Global fit of binding partners for all METRIS experiments performed.
(A) Global Log-Log plot showing linearity between rolling parameter and dissociation constant for all interactions measured. (B) Table of binding constants of tested interaction partners when …
Author response image 1
Distribution of rolling parameters for different PPI interactions.
Author response image 2
Comparison Between BLI and METRIS.
(A) Rolling parameter measurements for 3 mutants that bind ubiquitin. (B) Fit of METRIS to Kd values. (C) Kdvalues obtained from METRIS and BLI. (D) BLI data for each of the mutants.
Author response image 3
Rolling parameter of biotin-streptavidin interaction on avidin substrate (A) and a biotinylated supported lipid bilayer (B).
Author response image 4
Rolling parameter as a function of rotational frequency for interactions.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | BirA-GST-Orc1-BAH; Orc1-BAH | This Study | Strahl Lab, pGEX vector, Figure 2 | |
| Recombinant DNA reagent | BirA-GST-DIDO1-PHD; DIDO1-PHD | This Study | Strahl Lab, pGEX vector, Figure 3 | |
| Recombinant DNA reagent | N-cys Ubiquitin; ubiquitin | Kamadurai, Hari B et al. ‘Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex.’ Molecular cell vol. 36,6 (2009): 1095–102. | pGEX vector, | |
| Recombinant DNA reagent | Ube2D1 | DaRosa, Paul A et al. ‘A Bifunctional Role for the UHRF1 UBL Domain in the Control of Hemi-methylated DNA-Dependent Histone Ubiquitylation.’ Molecular cell vol. 72,4 (2018): 753–765.e6. | Pet15 vector, | |
| Recombinant DNA reagent | UHRF1-SRA | This study | Harrison Lab, MBP-pQ80L, Figure 4 | |
| Recombinant DNA reagent | N-cys-UHRF1-UBL W2V | This study | Harrison Lab, MBP-pQ80L, Figure 4 | |
| Recombinant DNA reagent | N-cys-UHRF1-UBL | This study | Harrison Lab, MBP-pQ80L, Figure 4 | |
| Software | Able Particle Tracker | Mu Labs | Full Version | http://apt.mulabs.com/ |
| Software | Mathematica | Steimel Labs | ||
| Software | OGG Video Converter | Ogg-converter.net | Version 6 | |
| Software | Pymol | Schrödinger | V2.4 | |
| Software | Prism | GraphPad | ||
| Other | Streptavidin Coated Ferromagnetic Beads | Spherotech | SVFM-100–4 | |
| Other | Avidin Coated Slides | ArrayIt | SMV | |
| Other | Histone peptide array | Petell, Christopher J et al. ‘Improved methods for the detection of histone interactions with peptide microarrays.’ Scientific reports vol. 9,1 6265. 18 Apr. 2019 | ||
| Antibody | Anti-GST (rabbit polyclonal) | Epicypher | 13–0022 | (1:1000) |
| Antibody | Anti-Rabbit AlexaFluor-647 (goat polyclonal) | Invitrogen | A21244 | (1:10,000) |
