Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature

Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10(−6) s(−1) at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10(4) and 10(6), for which EX(2) exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.