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

Area of Science:

• Protein Engineering and Design
• Biophysical Chemistry
• Membrane Biology

Background:

• Designed peptides with defined structures are crucial for membrane-associated biological processes.
• Heme proteins are versatile, inspiring designs for helical peptides; however, designed beta-sheet heme proteins are underexplored due to folding complexities.
• Naturally occurring beta-sheet proteins can be membrane-soluble and bind heme, indicating potential for designed analogs.

Purpose of the Study:

• To design and characterize novel membrane-soluble beta-sheet peptides capable of binding heme and di-heme.
• To investigate the structural basis and functional capabilities of these designed peptides in a membrane environment.
• To explore the potential of beta-sheet protein design for new applications in biocatalysis and energy conversion.

Main Methods:

• Design of four- and six-stranded beta-sheet peptides incorporating D-amino acid residues for beta-turn nucleation.
• Utilized bis-His coordination between antiparallel beta-strands for heme ligation.
• Employed beta- and omega-amino acids to optimize high-affinity heme binding pockets (nanomolar dissociation constants).

Main Results:

• Successfully designed and structurally characterized membrane-soluble beta-sheet peptides binding heme and di-heme.
• Demonstrated increasing heme binding affinity with omega-amino acid alkyl chain length, achieving nanomolar affinity.
• Observed cooperative di-heme binding in a six-stranded peptide and functional peroxidase activity and electron transfer with cytochrome c.

Conclusions:

• This study presents the first designed functional beta-sheet proteins in a membrane environment.
• The developed peptides expand the toolkit for creating novel heme proteins with tailored functions.
• The findings pave the way for engineering membrane-active proteins for diverse biotechnological applications.