Investigation of Biological Roles of Protein Kinetic Stability in Extremophiles

摘要

Nature optimizes the stability of proteins according to the structural and functional requirements to benefit the organism. Most proteins are marginally stable and remain in equilibrium with their partially and globally unfolded states to allow their functional regulation. However, certain proteins that need to be resilient, are trapped by a high energy barrier that prevents them from rapidly unfolding. These proteins are referred to as hyperstable or kinetically stable proteins (KSPs) and are usually resistant to degradation, misfolding and aggregation. Since KSPs have limited access to transiently unfolded states, they remain unaffected by detergents like sodium dodecyl sulfate (SDS). This property of SDS resistance has been utilized to develop a unique method, diagonal two-dimensional (D2D) SDS-PAGE, to isolate KSPs from other proteins in a complex protein mixture like cell lysate. The previous application of this method has yielded 50 proteins from E. coli, which have been further analyzed to deduce the preliminary structural and functional biases of protein kinetic stability. For understanding the biological roles of kinetic stability, other biological systems need to be explored to expand the database of KSPs. From literature and previous studies, kinetic stability appears to impart an evolutionary advantage to proteins that need to survive under harsh conditions. Therefore, this thesis has been designed to gain insight on how protein kinetic stability could be used by extremophilic organisms for survival in hostile environments.;The thermoacidophilic archaeon Sulfolobus acidocaldarius and the thermophilic bacterium, Thermus thermophilus were selected as two extremophiles for this study. Application of the D2D SDS-PAGE method followed by proteomics analysis resulted in the identification of 130 KSPs, 63 from S. acidocaldarius and 67 from T. thermophilus. An extensive functional analysis suggested that majority of the KSPs identified in S. acidocaldarius and T. thermophilus were involved in metabolism and energy production. Additionally, a bias towards enzymes was observed for the KSPs when compared to their respective proteomes, with oxidoreductases being over-represented and ligases under-represented. Both organisms also exhibited several KSPs involved in stress response pathways, which protects the cells from oxidative damage encountered by the organisms at high temperatures, in addition to other extracellular and cytosolic stresses. About 17% of the KSPs from S. acidocaldarius were found to be involved in biofilm formation and 12% of the KSPs from T. thermophilus participated in transport and localization.;For a more comprehensive analysis, and contrast with a mesophilic organism, the KSPs identified from the extremophiles were compared to the 50 KSPs previously identified from E. coli. Interestingly, only four of the 180 KSPs overlapped in all the three organisms. These proteins are involved in life-sustaining functions such as stress response (superoxide dismutase, thioredoxin reductase), translation (elongation factor thermo unstable) and metabolism (purine nucleoside phosphorylase), thus implying a potential role of kinetic stability in preserving or performing these protein functions. Under metabolism, which was the main biological pathway linked to the KSPs, carbohydrate metabolism was found to be the most common function among the KSPs of all organisms, while amino acid metabolism was more frequent in thermophiles than E. coli, putatively due to the nutrient-poor and hostile nature of their thermophilic environment.;Overall, certain biological functions, particularly metabolism and stress response, appear to have a greater necessity for protein kinetic stability. The identification of hyperstable proteins in different organisms could be beneficial in fundamental research to understand mechanisms of defense and survival in extreme conditions, as well as in biotechnology applications.