Molecular dynamics simulations at high temperatures of the Aeropyrum pernix L7Ae thermostable protein: Insight into the unfolding pathway

Most life forms on earth live at temperatures below 50 °C. Within these organisms are proteins that form the three-dimensional structures essential to their biological activity and function. However, some thermophilic life forms can resist higher temperatures and have corresponding adaptations to preserve protein function at these high temperatures. Among the structural factors responsible for this resistance of thermophilic proteins to high temperatures is the presence of additional hydrogen bonds in the thermophilic proteins, which means that the structure of the protein is more resistant to unfolding. Similarly, thermostable proteins are rich in structure-stabilizing salt bridges and/or disulfide bridges. In this context, we perform multiple replica molecular dynamics simulations at different temperatures on the Aeropyrum pernix (L7Ae) protein (from the crenarchaeal species A. pernix), known for its high melting temperature, and this in the aim to elucidate the structural factors responsible for its high thermostability. The results reveal that between the most sensitive regions of the protein to the increase of temperature are the loops L₁, and L₅, which surround the hydrophobic core region of the protein, besides the loop L₉, and the C-terminal α₅ region. This latter is the longer alpha helix of the protein secondary structure motifs and it is the first to be denaturated at 450 K, while the rest of the protein secondary structure motifs at this temperature were intact. The mechanism of unfolding that follows this protein at 550 K is similar to other thermophile proteins found in literature, with the opening of the loops that surround the hydrophobic core of the protein. So, the latter is completely exposed to the solvent, and partially denatured. The total denaturation process of the protein takes an average time of 40 ns to be achieved. Our investigation also shows that all the calculated salt bridges, with distances less than or equal to 6 A°, are on the periphery part of the protein, exposed to the solvent. However, the hydrophobic core of the protein is not involved in the formation of salt bridges, but rather with formation of some important hydrogen bondings that still persist even at 450 K. So, optimizing hydrogen bonding, near or within the core region, at high temperatures is a strategy that follows this thermostable protein to protect its hydrophobic core from denaturation, and ensure the thermal stability of the protein.