Rubredoxin from the hyperthermophile Pyrococcus furiosus (Pf Rd) is an extremely thermostable protein, which makes it an attractive subject of protein folding and stability studies. A fundamental question arises as to what the reason for such extreme stability is and how it can be elucidated from a complex set of interatomic interactions. We addressed this issue first theoretically through a computational analysis of the hydrophobic core of the protein and its mutants, including the interactions taking place inside the core. Here we show that a single mutation of one of phenylalanine's residues inside the protein's hydrophobic core results in a dramatic decrease in its thermal stability. The calculated unfolding Gibbs energy as well as the stabilization energy differences between a few core residues follows the same trend as the melting temperature of protein variants determined experimentally by microcalorimetry measurements. NMR spectroscopy experiments have shown that the only part of the protein affected by mutation is the reasonably rearranged hydrophobic core. It is hence concluded that stabilization energies, which are dominated by London dispersion, represent the main source of stability of this protein.
