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| Status |
Public on Jul 12, 2024 |
| Title |
MP3-seq: Massively parallel measurement of protein-protein interactions by sequencing |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
Abstract: Protein-protein interactions (PPIs) regulate many cellular processes, and engineered PPIs have cell and gene therapy applications. Here we introduce massively parallel protein-protein interaction measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast-two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs, and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Finally, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics simulation energy terms to predict MP3-seq values. We find that AF-M-based models could be valuable for pre-screening interactions, but that measuring interactions experimentally remains necessary to rank their strengths quantitatively.
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| Overall design |
The interactions of barcoded synthetic designed heterodimers were measured using a high throughput yeast two hybrid assay. Protein pairs were subject to an interaction dependent growth selection step. Expression plasmids were subsequently extracted and resulting barcodes were counted using an Illumina Next Seq (NGS) , and pre and post selection barcodes were processed using DeSeq2 to get a measure of PPI strength.
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| Contributor(s) |
Baryshev A, La Fleur A, Groves B, Michel C, Baker D, Ljubetič A, Seelig G |
| Citation(s) |
39192093 |
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| Submission date |
Jul 08, 2024 |
| Last update date |
Oct 21, 2024 |
| Contact name |
Alyssa La Fleur |
| Organization name |
University of Washington
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| Lab |
Seelig Lab
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| Street address |
Campus Box 352500 185 Stevens Way
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-2500 |
| Country |
USA |
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| Platforms (2) |
| GPL17143 |
Illumina MiSeq (Saccharomyces cerevisiae) |
| GPL26302 |
NextSeq 550 (Saccharomyces cerevisiae) |
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| Samples (36)
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| GSM8385373 |
DHD0 and DHD1 His- Barcodes Biol Rep 2 |
| GSM8385374 |
DHD0 (subset), DHD2, mALb (T) His+ |
| GSM8385375 |
DHD0 (subset), DHD2, mALb (T) His+- |
| GSM8385376 |
DHD0, DHD1, DHD2, mALb (T) His+ Biol Rep 1 |
| GSM8385377 |
DHD0, DHD1, DHD2, mALb (T) His- Biol Rep 1 |
| GSM8385378 |
Bcl & inhibitors His+ |
| GSM8385379 |
Bcl & inhibitors His- |
| GSM8385380 |
DHD0, DHD1, DHD2, mALb (T) His+ Biol Rep 2 |
| GSM8385381 |
DHD0, DHD1, DHD2, mALb (T) His- Biol Rep 2 |
| GSM8385382 |
NCIP His+ Biol Rep 1 24Hr Sample |
| GSM8385383 |
NCIP His+ Biol Rep 1 44Hr Sample |
| GSM8385384 |
NCIP His+ Biol Rep 1 60Hr Sample |
| GSM8385385 |
NCIP His- Biol Rep 1 (from 24 hr start) |
| GSM8385386 |
NCIP His- Biol Rep 1 (high 3-AT) (from 24 hr start) |
| GSM8385387 |
NCIP His+ Biol Rep 2 |
| GSM8385388 |
NCIP His- Biol Rep 2 |
| GSM8385389 |
NCIP His- (high 3-AT) |
| GSM8385390 |
Bcl & inhibitors His+ |
| GSM8385391 |
Bcl & inhibitors His- |
| GSM8385392 |
NICP, mALB (T) His+ |
| GSM8385393 |
NICP, mALB (T) His- |
| GSM8385394 |
An/Bn, NICP, N, PA series His+ |
| GSM8385395 |
An/Bn, NICP, N, PA series His- |
| GSM8385396 |
mALB (R) His+ |
| GSM8385397 |
mALB (R) His- |
| GSM8385398 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His+ Biol Rep 1 |
| GSM8385399 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His+ Biol Rep 1 (technical replicate) |
| GSM8385400 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His- Biol Rep 1 |
| GSM8385401 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His- Biol Rep 1 (technical replicate) |
| GSM8385402 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His+ Biol Rep 2 |
| GSM8385403 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His+ Biol Rep 2 (technical replicate) |
| GSM8385404 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His- Biol Rep 2 |
| GSM8385405 |
NICP, mALB (T), mALb (R),An/Bn, P, N, PA series His- Biol Rep 2 (technical replicate) |
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| Relations |
| BioProject |
PRJNA1133167 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE271790_RAW.tar |
1.2 Mb |
(http)(custom) |
TAR (of CSV) |
| GSE271790_barcodes_to_proteins_all_experiments.xlsx |
47.7 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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