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

Abstract

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 Ϸmillisecond 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 EX2 exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal͞mol 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.

Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules

“…These results suggest that the regions around those atoms are relatively inaccessible to solvent. The conclusion from these H͞D exchange ratio measurements is roughly comparable with the results from NMR studies (52,53), which indicate that there are two broad regions in the protein that display slow NMR H͞D exchange behavior, one centered around the Cys-5͞Cys-8 region and the other around Cys-38͞Cys-41 (Fig. 2b Bottom).…”

Protein stabilization by compatible solutes

“…The EX2 exchange regime has been established in various rubredoxins (as in most globular stable proteins) [27,42]. In fact, EX1 reactions are rarely seen in stable proteins, occurring mostly under the conditions used in some protein refolding experiments [43][44][45][46].…”

“…In fact, this rigidification has been revealed by H-D exchange experiments [23,26]. However, this view was recently challenged by the observation of relatively fast exchange rates in the rubredoxin from Pyrococcus furiosus, the most stable protein known to date [27]. In this context, assessing the changes in the dynamic behaviour of proteins in the presence of solutes is expected to shed light on the stabilization phenomenon.…”

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

“…Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3, 4). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity.Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4, 5), whereas others, on ␣-amylase (6) and on rubredoxin (7,8), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects.…”