Sarkosyl: A milder detergent than SDS for identifying proteins with moderately high hyperstability using gel electrophoresis.

Sodium dodecyl sulfate (SDS) is a detergent used as a strong denaturant of proteins in gel electrophoresis. It has previously been shown that certain hyperstable, also known as kinetically stable, proteins are resistant to SDS and thus require heating for their denaturation in the presence of SDS. Because of its high denaturing strength, relatively few proteins are resistant to SDS thereby limiting the current use of SDS-PAGE for identifying hyperstable degradation-resistant proteins. In this study, we show that sarkosyl, a milder detergent than SDS, is able to identify proteins with moderately high kinetic stability that lack SDS-resistance. Our assay involves running and subsequently comparing boiled and unheated protein samples containing sarkosyl, instead of SDS, on PAGE gels and identifying subsequent differences in protein migration. Our results also show that sarkosyl and SDS may be combined in PAGE experiments at varying relative percentages to obtain semi-quantitative information about a protein's kinetic stability in a range inaccessible by probing through native- or SDS-PAGE. Using protein extracts from various legumes as model systems, we detected proteins with a range of protein stability from nearly SDS-resistant to barely sarkosyl resistant.

Biological Roles of Protein Kinetic Stability.

A protein's stability may range from nonexistent, as in the case of intrinsically disordered proteins, to very high, as indicated by a protein's resistance to degradation, even under relatively harsh conditions. The stability of this latter group is usually under kinetic control because of a high activation energy for unfolding that virtually traps the protein in a specific conformation, thereby conferring resistance to proteolytic degradation and misfolding aggregation. The usual outcome of kinetic stability is a longer protein half-life. Thus, the protective role of protein kinetic stability is often appreciated, but relatively little is known about the extent of biological roles related to this property. In this Perspective, we will discuss several known or putative biological roles of protein kinetic stability, including protection from stressors to avoid aggregation or premature degradation, achieving long-term phenotypic change, and regulating cellular processes by controlling the trigger and timing of molecular motion. The picture that emerges from this analysis is that protein kinetic stability is involved in a myriad of known and yet to be discovered biological functions via its ability to confer degradation resistance and control the timing, extent, and permanency of molecular motion.

Analyzing bean extracts using time-dependent SDS trapping to quantify the kinetic stability of phaseolin proteins.

In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60-75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20-100 years at 24 °C and 2-7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification.