Thermostabilization mechanisms in thermophilic versus mesophilic three-helix bundle proteins.

The engineered three-helix bundle, UVF, is thermostabilized entropically due to heightened, native-state dynamics. However, it is unclear whether this thermostabilization strategy is observed in natural proteins from thermophiles. We performed all-atom, explicit solvent molecular dynamics simulations of two three-helix bundles from thermophilic H. butylicus (2lvsN and 2lvsC) and compared their dynamics to a mesophilic three-helix bundle, the Engrailed homeodomain (EnHD). Like UVF, 2lvsC had heightened native dynamics, which it maintained without unfolding at 100°C. Shortening and rigidification of loops in 2lvsN and 2lvsC and increased surface hydrogen bonds in 2lvsN were observed, as is common in thermophilic proteins. A buried disulfide and salt bridge in 2lvsN and 2lvsC, respectively, provided some stabilization, and addition of a homologous disulfide bond in EnHD slowed unfolding. The transferability and commonality of stabilization strategies among members of the three-helix bundle fold suggest that these strategies may be general and deployable in designing thermostable proteins.

Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin.

The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(δ) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(α) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.