Applications of gel electrophoresis to study kinetically stable proteins in mushrooms and sesame seeds
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Authors
Shah, Isha
Issue Date
2025-05
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemistry
Alternative Title
Abstract
Kinetically stable proteins (KSPs) are a special class of proteins that possess a high activation energy barrier for unfolding and thus exhibit remarkable resistance to denaturation. This stability allows these proteins to persist for longer periods of time in harsh in vivo conditions, protecting them from irreversible inactivation by aggregation or proteolysis. The correlation between protein kinetic stability (KS) and resistance to denaturation by the surfactant sodium dodecyl sulfate (SDS) has been well documented. This has led to the use of SDS-resistance as a probe for KS. Several polyacrylamide gel electrophoresis (PAGE) based assays have been developed to identify KS in protein isolates and complex lysates. This study involved the development of an aqueous extraction method for mushrooms, an organism of much interest for potential applications in mycoremediation, biofuel production, and the medicinal field. Mushrooms have a variety of enzymes that are excreted to break down nutrients found in the surrounding environment into compounds that can be readily absorbed by the mycelia. Due to the excretory nature of these proteins, it was hypothesized that mushrooms would possess many proteins with high KS to maintain their function in a variety of pHs, temperatures, and humidity levels. Here, the KS of proteins from several edible mushrooms (white button, cremini, portabella, enoki, shiitake, lion mane, king oyster, and oyster) was probed by diagonal two-dimensional (D2D) SDS-PAGE, which exploits the SDS-resistance of KSPs. This study revealed significant variations in KSP abundance, with white button and oyster mushrooms exhibiting particularly high amounts. Conversely, lion's mane displayed a lower abundance, demonstrating the species-specific nature of KSPs. The gels also highlighted the potential influences from disulfide bonds, phenolic compounds, and protease activity. Oyster and white button mushroom were furthered studied to characterize the subproteome of their KSPs.
In total, sixty-three KSPs and fifty-six KSPs were identified in oyster and white button mushroom, respectively. Approximately half of the KSPs were hydrolytic enzymes, including glycoside hydrolases and proteases. Additional KSPs that were identified were those that functioned in a bioprotective or regulatory role. Structural analysis, aided by AlphaFold, the CATH database, and homology studies, highlighted the prevalence of complex secondary structures, particularly α/β folds, and quaternary structures.
Active site alignments of kinetically stable polyphenol oxidases and hydrolases with current enzymes suggest a potential application of higher-order fungi for mycoremediation, medicinal applications, and biofuel production. The isolation and identification of these KSPs is one of the first studies focusing on the potential applications for individual proteins in higher-order fungi, rather than the application of the whole fruiting body and provided a promising foundation for enzyme activity studies as well as methods needed to probe other mushrooms and fungi.
Furthermore, the study explored the impact of post-translational modifications (PTMs), specifically glycosylation and coordinated metals, on protein stability in mushrooms. It demonstrated that glycosylation and metalation play key roles in deglycosylation using PNGase F resulted in a reduction of KSP abundance, particularly in white button mushrooms, highlighting the crucial role of glycans in maintaining structural integrity. Metal removal using EDTA also resulted in a decrease in KSP abundance, confirming the importance of metal ions in protein stability. This research underscored the untapped potential of edible mushrooms as a source of KSPs for various applications. Future studies should focus on elucidating structure-function relationships, investigating PTMs, and conducting activity assays of individual KSPs.
An additional study focused on probing the KS of allergenic proteins in the newest major allergen, sesame seeds, as highly stable proteins have often been linked to food allergies. In order to assess proteins that may possess a range of KS, a mild surfactant, sodium lauroyl sarcosinate (SAR), was used as a proxy for an intermediate level of KS. Two major allergens were identified to be kinetically stable: the 2S albumin (Ses i 1) and the 11S globulin (Ses i 6 and Ses i 7). A semi-quantitative comparison of their KS was also assessed using various SDS:SAR ratios, and their KS was then compared to their allergenic prevalence to assess their relative importance in eliciting allergy. A correlation was observed between the allergenic potential of the protein and the KS. This study demonstrated that KS could be a useful tool to probe the potential allergenicity of food proteins. The observed correlation between high stability and allergenic potency highlights the need for further investigations into the structural and functional properties of these proteins, with implications for mitigating sesame allergies and improving food safety.
Description
May2025
School of Science
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY