Beyond sds: expanding the surfactant toolkit for protein kinetic stability
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Authors
Rugaber, Evelyn-Grace
Issue Date
2025-10
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemistry
Alternative Title
Abstract
Kinetically stable proteins (KSPs) are proteins defined by their significant energy barrier to unfolding, which kinetically traps them in their native conformation. KSPs have extraordinarily long half-lives and resistance to proteolysis, misfolding, and aggregation even in harsh conditions such as high temperatures and extreme pH. KSPs are also resistant to denaturation by surfactants such as SDS, which makes SDS-resistance a highly effective probe for high KSPs. However, because SDS is such a strong surfactant, SDS-based probes for KS exclude proteins that possess a significant level of KS yet are below the threshold of SDS-resistance. Sarkosyl (SAR), a weak surfactant, has been used to identify these proteins and expand the spectrum of KS that can be assessed with surfactant-based probes. This thesis focuses on exploring the mechanism by which surfactant-based probes identify different levels of KS and their application to investigate KS within the model system of serine proteases. While SAR can probe for moderate KS, the mechanism by which it does so is not well understood. This study examines the mechanistic differences between SAR and SDS and evaluates the validity of using SAR as a quantitative probe for KS. Results from a Surfactant-Release assay suggest that the binding and trapping of the protein unfolded state is essentially irreversible for both SDS and SAR. However, results from a SAR-TraP assay, which monitors protein unfolding kinetics via the loss of SAR-resistance over time, shows inconsistent rates of trapping compared to the known unfolding rate of the proteins. The slower rate of trapping by SAR for CHT in particular is consistent with the frequent aggregation and smearing in SAR-PAGE not seen in SDS-PAGE. This strongly suggests that SAR cannot compete against protein-protein interactions effectively. Overall, the results suggest that the greater denaturing strength of SDS compared to SAR is not caused by differences in their binding-trapping of the denatured state or the subsequent rate of release; instead, it appears to be due to SDS’s ability to interact with the native state of proteins with moderate or lower KS to cause their unfolding. Ultimately, the use of SAR as a probe for moderate KS is supported, though SAR cannot be used for reliable quantitative measures of protein KS.
Alternative weak surfactants to SAR may similarly probe for moderate KS at varying levels while providing higher quality data. Four anionic surfactants (sodium lauryl ether sulfate (SLES), sodium dodecylbenzene sulfonate (SDBS), disodium lauryl sulfosuccinate (DLSS), and sodium lauryl sulfoacetate (SLSa)) were explored for this purpose, with their denaturation potency and compatibility with gel-based methods assessed. The surfactants were all successful in identifying proteins with moderate KS and exhibited varying denaturation strengths following the ranking: SDS > SDBS ≈ SLSa > SLES > SAR ≈ DLSS. These results indicate that using different surfactants with varying denaturing capacities may expand the spectrum of KS that can be assessed and provide at least semi-quantitative information on protein KS.
Finally, a model system of small, monomeric serine proteases was reassessed to explore KS as a critical factor in protease function, regulation, and persistence. SDS- and SAR-resistance were used to identify low, moderate, or high KS in S1 and S8 protease families. Bioinformatic, structural, and environmental characteristics were then assessed and correlated with KS. Results of these analyses reveal links between KS and topological complexity, and the possession of large pro-regions involved in protein folding. The observation that mammalian proteases possess moderate-low KS, whereas the bacterial proteins have high KS, suggests that KS plays a role in balancing persistence and regulation of proteases in vivo. These findings highlight the relationship of structure and evolutionary pressures with protein KS and provide a basis for future studies focusing on the structural basis of KS in a broader context.
Description
October2025
School of Science
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY