Metalloprotein engineering involves the modification of the scaffold or metal-binding site of an existing metalloprotein or the introduction of a new metal-binding site within a protein. Metalloprotein engineering is used to study structure-function relationships, enhance the properties of a metalloprotein, or design functions beyond those observed in naturally-occurring metalloproteins. Of particular interest are metalloproteins with metal clusters, which are known to catalyze multi-electron redox reactions such as the four-electron oxidation of water to O2 or the reverse, reduction of O2 to H2O. For example, synthetic stacked cobalt porphyrins have been shown to catalyze the O2 reduction reaction almost exclusively to water, a crucial reaction to most fuel cells. Based on protein structure analysis and modeling, heme c insertion into the protein CHIP is hypothesized to generate a dimer with a stacked heme c structure, not yet seen in naturally-occurring proteins. To test the hypothesis of stacked hemes c, two expression vectors were created using seamless ligation cloning extract (SLiCE): one for wild-type CHIP and one where a heme c-binding CXXCH motif has been introduced into the sequence (CHIP-heme). Wild-type CHIP expresses in auto-inducing media, terrific broth, and LB broth. However, holo-CHIP-heme was only expressed inconsistently in auto-inducing media. Based on SDS-PAGE analysis comparing osmotic shock periplasmic extraction to complete cell lysis, it is hypothesized that CHIP-heme expression is not robust due to poor periplasmic secretion, which is signaled using the N-terminal pelB signal peptide. The secretion is necessary for heme attachment via the cytochrome c maturation (Ccm) system. Three new CHIP-heme vectors are currently being constructed with new signaling sequences: OmpA, Nfra, and a modified PelB signal. Each gene will be expressed and the protein product tested for successful heme attachment.

Template-based engineering of stacked c-type hemes

2019 Spring National Meeting

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