Proteomics-Level Identification of Degradation-Resistant Proteins Provide Insight about their Potential Roles in Organismal Adaptation to Stress

Abstract

Some proteins are hyperstable, as demonstrated by their resistance to proteolysis and detergents, and their long half-life. Some of these proteins are characterized by having a high energy barrier toward unfolding that virtually traps them in their native state. This property of proteins, known as “kinetic stability” (KS), appears to be a strategy used by “mother nature” to protect certain proteins against aggregation, and premature degradation under stress conditions. In addition to saving organismal resources by avoiding frequent degradation-biosynthesis of proteins, KSPs could function under extreme stress to maintain vital functions needed for survival. On the basis of our observed correlation between KS and a protein's resistance to the detergent SDS, we developed a diagonal two-dimensional (D2D) SDS-PAGE method for the proteome-level identification of KSPs. We applied D2D SDS-PAGE to the lysate of Thermus thermophilus and E. coli followed by proteomics analysis, and identified over 100 KSPs. We also analyzed the cell lysates of various prokaryotic and eukaryotic organisms. Our results suggest that KS was likely a critically important property of proteins for the adaptation and survival of microbial organisms under stress conditions. In contrast, the minimal abundance of KSPs in eukaryotic organisms implies an evolutionary compromise of KS in favor of more complex cellular defense, function, and regulation. Thus, D2D SDS-PAGE is a simple and powerful method that may be applied to any complex mixture of proteins to explore the biological and pathological significance of protein kinetic stability.