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The field of de novo protein design has met with considerable success over the past few decades. Heme, a cofactor has often been introduced to impart a diverse array of functions to a protein, ranging from electron transport to respiration. In nature, heme is found to occur predominantly in α-helical structures over β-sheets, which has resulted in significant designs of heme-proteins utilizing coiled coil helices. By contrast, there are only a few known β-sheet proteins that bind heme and designs of β-sheets frequently result in amyloid-like aggregates. This review reflects on our success with designing a series of multi-stranded β-sheet heme binding peptides that are well folded both in aqueous and membrane-like environments. Initially, we designed a β-hairpin peptide that self-assembles to bind heme and performs peroxidase activity in membrane. The β-hairpin was optimized further to accommodate a heme binding pocket within multi-stranded β-sheets for catalysis and electron transfer in membranes. Furthermore, we de novo designed and characterized β-sheet peptides and mini-proteins soluble in aqueous environment capable of binding single and multiple hemes with high affinity and stability. Collectively, these studies highlight substantial progress made towards the design of functional β-sheets. Ministry of Education (MOE) Accepted version AS would like to thank Ministry of Education, Singapore and Nanyang Technological University for Graduate Research Scholarship.

E. coli and Salmonella are two of the most common bacterial pathogens involved in food- and water-borne-related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFUmL1 of E. coli in water within 6 hours by targeting the bacterias surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants. Ministry of Education (MOE) Accepted version This work was funded by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-1-025) and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14.