Metalloproteins accomplish a broad range of crucial functions in biological systems. The ability to design metalloproteins with customized binding or catalytic properties could have a wide variety of applications in the fields of medicine, chemical synthesis or energy production. One common way to alter the function and properties by the re-design of existing metalloproteins is to substitute one metal for another. Presented here is progress toward the metal-substitution of two metalloprotein templates: (1) A genetically-encoded calcium indicator, Twitch 2B, and (2) mitochondrial cytochrome c. The progress toward changing the metal specificity of Twitch-2B focused on using Polymerase Chain Reaction (PCR) methods and Seamless Ligation Cloning Extract (SLiCE) cloning to simultaneously and combinatorially mutate eight different amino acids located on the DF-hand motif of Troponin C, the calcium-binding region of Twitch-2B. We discovered that touchdown and combinatorial PCR methods generated the desired product. . The metal-substitution of the heme iron in cytochrome c with cobalt has been previously performed chemically to yield a hydrogen evolution catalyst, demonstrating that the direct expression of cobalt-substituted hemes would be desirable. Our approach involved mutation of holocytochrome c synthase, the enzyme responsible for the covalent attachment of heme to cytochrome c’s Cys-X-X-Cys-His motif. The results demonstrate that E. coli could express cytochrome c in minimal medium with elevated [Co<sup>2+</sup>] by gradually ramping up the cobalt concentration over a period of two weeks. Preliminary results possibly demonstrate a mixture of cobalt and native-iron cytochrome c.

Metal-substitution using two metalloprotein design templates: Genetically encoded calcium indicator and mitochondrial cytochrome <i>c</i>

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.

Metal-substitution using two metalloprotein design templates: Genetically encoded calcium indicator and mitochondrial cytochrome c