
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.05.611266
preprint
1
Article
Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli
Treviño Matthew A. http://orcid.org/0009-0009-1688-3075

Amankwah Kofi
Fernandez Daniel http://orcid.org/0000-0002-6221-152X

Weston Scott
Stewart Claire J. http://orcid.org/0000-0002-3071-3322

Gallardo Jaime Morales
Shahgholi Mona http://orcid.org/0000-0002-8879-4305

Sharaf Naima G. http://orcid.org/0000-0002-3662-9228

07 9 2024
2024.09.05.611266https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.05.611266
nihpp-2024.09.05.611266.pdf
Abstract

YcjN is a putative substrate-binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli . Here, we determine the crystal structure of YcjN to a resolution of 1.95 Å, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.
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pmc
