30 Fendos and Engelman: pHLIP and acidity as universal biomarker for cancer Figure 1. A) pHLIP exists in three biophysical states depending on context: State I, as a largely unstructured monomer in aqueous solution at slightly basic or neutral pH; State II, as a largely unstructured monomer partitioned to the membrane surface in the presence of li- posomes; and State III, as an inserted, bilayer-spanning α-helix at low pH. All states are re- versible as denoted by the double arrows. Adapted from Biophys J. 2007;93(7):2363-72 [17]. B) The primary sequence of pHLIP. r ed indicates hydrophobic residues, black indicates polar residues, blue indicates acidic residues, and underlined green indicates basic residues. The bracket denotes residues at positions 9-30 containing the two aspartic acids (blue D) at po- sitions 14 and 25. The highlighted yellow residues comprise the cysteine tag attached to the inserting end of the peptide for use in conjugating cargo molecules for translocation into cells. C) The mechanism of pHLIP insertion at low pH: Insertion is largely dependent on the titra- tion of the two aspartic acids (blue circles) at positions 14 and 25. Acidic protons (red circles) bind to the basic aspartic side chains at low pH, neutralizing the charge and converting the side chains into a more hydrophobic form. This thermodynamic conversion facilitates inser- tion by lowering the energy barrier (by about 3 kc al/mol [13]) for stable interaction with the membrane bilayer. at best, a minimal subset of cancer types. De- spite more than a half century of intense can- cer research, we still have not found a protein or nucleic acid that functions as a “universal” biomarker for all cancers. Interestingly, there is a universal marker for virtually all solid tumors often over- looked and lost in the vast wealth of cancer literature. This universal marker is localized cellular acidity [4]. It has long been ob- served that cancer tissues in vivo are more acidic than normal tissues. This observation is attributed to the effects of hypoxia and called the Warburg effect [5] and from the heightened metabolic activity of cancer cells. A variety of conjectures and observa- tions exist in the literature about how this lo- calized acidity may facilitate certain cancer traits such as longevity, growth, and metas- tasis [6,7,8]. Several methods, including magnetic resonance imaging [9] and direct in vivo measurements with glass probes