School of Integrative Biological & Chemical Sciences Faculty Publications and Presentations
Document Type
Article
Publication Date
12-2024
Abstract
The late embryogenesis abundant proteins (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs’ presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high-throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire subpopulation of phage retained by each LEAP ortholog. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (paired-end phage sequencing) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two dehydrin orthologs (LEA14 and GmPm12) compared to bovine serum albumin (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature-related intensity change analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation assays. The results provide insights into the involvement of the dehydrin Y-segments and K-domains in protein binding.
Recommended Citation
Unêda-Trevisoli, Sandra Helena, Lynnette MA Dirk, Francisco Elder Carlos Bezerra Pereira, Manohar Chakrabarti, Guijie Hao, James M. Campbell, Sai Deepshikha Bassetti Nayakwadi et al. "Dehydrin Client Proteins Identified Using Phage Display Affinity Selected Libraries Processed With Paired-End Phage Sequencing." Molecular & Cellular Proteomics 23, no. 12 (2024): 100867. https://doi.org/10.1016/j.mcpro.2024.100867
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Publication Title
Molecular & Cellular Proteomics
DOI
10.1016/j.mcpro.2024.100867
Comments
http://creativecommons.org/licenses/by/4.0/