Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'.

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.