Beyond MALDI: Molecular recognition of gentisic acid by a model protein
Ifeagwu, Marvin
Ifeagwu, Marvin
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Publication Date
2025-01-22
Type
master thesis
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Abstract
The benzoic acid derivative gentisic acid (2,5-dihydroxybenzoic acid), is a common sample matrix molecule used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of proteins. Beyond its use in MALDI, gentisic acid has been shown to exhibit notable polymorphic properties i.e., capable of adopting more than one crystal form under varying crystallization conditions.
In this study, we explored the co-crystallization of gentisic acid with lysozyme, a well characterized model protein known for its ease of crystallization. Motivation for this work stems from the hypothesis that the polymorphic nature of gentisic acid, may play a role in driving and/or directing protein assembly, potentially resulting in novel crystalline architectures of lysozyme.
X-ray crystallographic analysis revealed two distinct binding modes of gentisic acid on the protein surface. These binding modes show gentisic acid engaging in hydrogen bonding, salt bridge and cation-π interactions with residues on the protein surface. Additionally, a ligand-free structure of lysozyme, devoid of gentisic acid was obtained in the presence of Mg²⁺. This result provides some insight into metal coordination and its impact on the protein binding potential of simple ligands such as gentisic acid.
We also investigated co-crystallization of the cationic macrocycle, Bluebox with the neutral model protein Ralstonia solanacearum lectin (RSL), in an attempt to investigate molecular recognition using cationic receptors. However, our attempts yielded two small molecule crystals of Bluebox–Acetate and Bluebox–SCN salts. The host-guest properties of Bluebox may warrant further exploration in protein co-crystallization.
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Publisher
University of Galway
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Attribution-NonCommercial-NoDerivatives 4.0 International