Designed multi-stranded heme binding β-sheet peptides in membrane.

Designed peptides demonstrating well-defined structures and functioning in membrane environment are of significant interest in developing novel proteins for membrane active biological processes including enzymes, electron transfer, ion channels and energy conversion. Heme proteins' ability to carry out multiple functions in nature has inspired the design of several helical heme binding peptides and proteins soluble in water and also recently in membrane. Naturally occurring β-sheet proteins are both water and membrane soluble, and are known to bind heme, however, designed heme binding β-sheet proteins are yet to be reported, plausibly because of the complex folding and difficulty in introducing heme binding sites in the β-sheet structures. Here, we describe the design, NMR structures and biochemical functional characterization of four stranded and six stranded membrane soluble β-sheet peptides that bind heme and di-heme, respectively. The designed peptides contain either (D)P-G or (D)P-(D)A residues for the nucleation of β-turns intended to stabilize multi-stranded β-sheet topologies and ligate heme with bis-His coordination between adjacent antiparallel β-strands. Furthermore, we have optimized a high affinity heme binding pocket, K(d) ∼ nM range, in the adjacent β-strands by utilizing a series of four stranded β-sheet peptides employing β- and ω-amino acids. We find that there is a progressive increase in cofactor binding affinity in the designed peptides with the alkyl chain length of ω-amino acids. Notably, the six stranded β-sheet peptide binds two molecules of heme in a cooperative fashion. The designed peptides perform peroxidase activity with varying ability and efficiently carried out electron transfer with membrane associated protein cytochrome c. The current study demonstrates the designing of functional β-sheet proteins in a membrane environment and expands the repertoire of heme protein design.