Proposal: pHLIP generation in E.coli By: Brandon Lee and Ahad Waraich

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

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

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.

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 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)

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

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?

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

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