title: Constructing a man-made c-type cytochrome maquettein vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.
abstract: The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to producein vivoan artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance inde novoprotein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteinsin vivo.
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.|The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: J L Ross
authors.initials: JLR
authors.lastname: Anderson
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors.forename: Craig T
authors.initials: CT
authors.lastname: Armstrong
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: Goutham
authors.initials: G
authors.lastname: Kodali
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Lichtenstein
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Watkins
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Mancini
authors.affiliation: School of Chemistry, University of Bristol, Bristol, BS8 1TS, UK.
authors.forename: Aimee L
authors.initials: AL
authors.lastname: Boyle
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: Tammer A
authors.initials: TA
authors.lastname: Farid
authors.affiliation: School of Chemistry, University of Bristol, Bristol, BS8 1TS, UK.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Moser
authors.affiliation: The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.
authors.forename: P Leslie
authors.initials: PL
authors.lastname: Dutton