The hydrophobicity of peptides limits their availability to bind to interacting domains
- 1Université Laval
Abstract
A challenge in synthetic biology is to transfer the knowledge acquired in vitro to in vivo applications. For instance, binding affinity between peptide binding domains (PBDs) and their binding peptides is usually measured in simple buffers and in the absence of the cell machinery responsible for synthesis and proteostasis. Consequently, there is often a discrepancy between interaction quantified in vitro and in vivo. To identify strongly binding PBD-peptide pairs for in vivo applications, we aim to better understand the determinants of synthetic interaction in living yeast cells. We designed libraries of peptides expected to bind to PBDs based on data collected by phage-display. Using an in vivo protein interaction assay, we assessed the interaction strength of >6,500 PBD-peptide variants in yeast. We found that most substitutions disrupt binding, but that 3.4% increased binding strength to levels higher than expected from the phage-display data. We asked if, by using our mutational data, it was possible to optimize peptide sequences to gain even more binding strength and show additivity effect up to four substitutions. We found that PBD specificity is consistent between in vitro and in vivo assays. We hypothesized that in vivo peptide abundance could contribute to the discrepancy between the PCA and the phage-display results. We performed assays to detect in vivo peptide availability, reporting on peptide abundance, and we found that peptides with a stronger binding also show a higher availability in cells. We show that peptide availability, which seems mainly defined by peptide hydrophobicity, is key for the design of strong synthetic interactions.
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BibTeX
@misc{lemieux2026hydrophobicity,
title = {{The hydrophobicity of peptides limits their availability to bind to interacting domains}},
author = {P. Lemieux and X. Barbeau and S. Dibyachintan and A. K. Dube and P. Lague and C. R. Landry},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.752983},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.752983v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - The hydrophobicity of peptides limits their availability to bind to interacting domains
AU - P. Lemieux
AU - X. Barbeau
AU - S. Dibyachintan
AU - A. K. Dube
AU - P. Lague
AU - C. R. Landry
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.752983
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.752983v1
AB - A challenge in synthetic biology is to transfer the knowledge acquired in vitro to in vivo applications. For instance, binding affinity between peptide binding domains (PBDs) and their binding peptides is usually measured in simple buffers and in the absence of the cell machinery responsible for synthesis and proteostasis. Consequently, there is often a discrepancy between interaction quantified in vitro and in vivo. To identify strongly binding PBD-peptide pairs for in vivo applications, we aim to better understand the determinants of synthetic interaction in living yeast cells. We designed libraries of peptides expected to bind to PBDs based on data collected by phage-display. Using an in vivo protein interaction assay, we assessed the interaction strength of >6,500 PBD-peptide variants in yeast. We found that most substitutions disrupt binding, but that 3.4% increased binding strength to levels higher than expected from the phage-display data. We asked if, by using our mutational data, it was possible to optimize peptide sequences to gain even more binding strength and show additivity effect up to four substitutions. We found that PBD specificity is consistent between in vitro and in vivo assays. We hypothesized that in vivo peptide abundance could contribute to the discrepancy between the PCA and the phage-display results. We performed assays to detect in vivo peptide availability, reporting on peptide abundance, and we found that peptides with a stronger binding also show a higher availability in cells. We show that peptide availability, which seems mainly defined by peptide hydrophobicity, is key for the design of strong synthetic interactions.
ER -