Abstract
Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers.
Highlights
Hyperthermostable enzymes are usually isolated from hyperthermophiles grown at extremely high temperatures [1]
Oligomerization of tcSOD We evaluated the oligomeric equilibrium of tcSOD by size-exclusion chromatography (SEC) and spectroscopic analysis by varying protein concentrations
It is of great interest to investigate the molecular mechanism for hyperthermostable proteins to fight with the off-pathway processes under the extreme high temperature
Summary
Hyperthermostable enzymes are usually isolated from hyperthermophiles grown at extremely high temperatures [1]. Hyperthermostable enzymes have very similar primary sequences and tertiary structures to their mesophilic homologues, they show remarkable stability against denaturation induced by heat or chemical denaturants [2]. Many structural stabilizing factors have been proposed to contribute to the extreme thermostability of soluble hyperthermostable proteins, such as stronger ion-pairing network, additional disulfide bridges, higher order oligomerization, conformational rigidity, fewer cavities and more compact structures when compared to the mesophilic proteins [1,2,3,4,5,6,7]. The characterization of the molecular origin of protein hyperthermostability can facilitate the applications of important industrial enzymes, and help us to understand the key factors in enzyme evolution and protein stability
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