Proposal: pHLIP generation in E.coli

1. Proposal: pHLIP generation in E.coli By: Brandon Lee and AhadWaraich

2. A peptide found in Bacteriorhodopsin. • pHLIP = pH Low-insertion peptide molecule • Sequence: AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG • - At low pH environments, spontaneous insertion (into membrane bilayers) and formation of transbilayer alpha-helices occurs. What is pHLIP?

3. A Bit of History and Why we should care • pHLIP first discovered by the Donald M. Engelman group at Yale University and collaborating members from the University of Rhode Island. • High extracellular acidity is associated with many pathological conditions: drug delivery and cell imaging can be done via pHLIP! High acidity is not observed in normal cellular environments • - tumors, • - infarcts, • - stroke-afflicted tissue, • - atherosclerotic lesions

4. How it works: What happens • Forms transmembrane alpha-helix: becomes rigid at low pH like nano-syringe • Inserts in low pH environments but only associates at pH > 7.0; Triggered by the increase of the peptide hydrophobicity due to the protonation of Asp residues induced by low pH. • N terminus stays outside, C terminus of the peptide is translocated across the bilayer • - Previous experiments show that disulfide bonding to the c-terminus can be cleaved by reducing environment within cell. Can attach dye’s or other molecules to this end.

5. Results of Several Studies Done • The delivery of phalloidin into cells by pHLIP. (a) Fluorescence images of HeLa cells incubated (for 1 h) with a pHLIP–S–S–Ph–TRITC cleavable construct • (2 M) at pH 7.4 • - Strong fluorescence of actin filaments was observed after pH 6.5 • incubation. Tumor and inflammation imaging: - (b) Overlay of pHLIP-Cy5.5 fluorescence and light images of mice bearing a tumor (7mmin diameter, 12 d after 106 cell implant)

6. What remains to be known? • The size of molecules that can be attached to pHLIP remains to be known • Polarity of molecules that can be attached also remains to be studied • What available drugs and imaging techniques can we apply to this molecule? • Overall, this molecule has not been explored in too much detail

7. What can we do? • Recent experiments by the Engelman group have shown that near-infrared fluorescence imaging works in mouse cancers and rat inflammatory arthiritis models using pHLIP as a delivery device • NIR fluorescent dyes can be conjugated with Cys or Lys residues placed on pHLIP N-terminus (outside of cell upon insertion). • - Dyes that can be used • - Cy5.5 or Alexa750 • Possibly initiatives: • Since NIR range (700 – 900 nm) can propagate through tissues in organs and whole bodies, why not use this for early tumor detection? • Another direction: Is there some way to make this delivery device easily accessible? Produced in large quantities?

8. Why Synthetic Biology? • pHLIP related to a peptide in bacteriorhodopsin found in halobacteria (not a model organism) • pHLIP has been created through solid-phase peptide synthesis (SPPS) via 9-fluorenylmethyloxycarbonyl (FMOC) chemistry, purified via reverse-phase chromatography • FMOC solid-phase peptide synthesis: Treat small, solid beads with linkers (functional units) that can be used to make peptide chains

9. Why Synthetic Biology? • SPPS may be “limited by yield” • Perhaps synthetic biology (generate it in E. coli, etc.) could be better • Even if there are no apparent problems with SPPS, there is no harm in trying to create the protein in a new way (like synthetic biology, with E. coli); something advantageous about a synthetic biology method could arise