The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7. change in acidity even before insertion. Localized extracellular acidity in solid tumours may be exploited for cancer diagnosis and treatment1,2,3,4. To this end, the 36-residue pH-low insertion peptide (pHLIP, with the sequence GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT) has been developed as imaging tools and carriers of therapeutic agents5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. The tumour-targeting ability of pHLIP is thought to be based on its insertion into membrane in response to environmental acidity8,21,22,23,24. Similarly, pHLIP can detect other pathological acidic microenvironments thioredoxin is shifted to 7.3C7.5 due to the formation of a water-mediated hydrogen bond51. A more nonpolar environment, that is, the one with lower dielectric constant than water (such as the membrane interiors), should also favour the formation of the neutral carboxylic acid, effectively increasing the pKa of D14/D25 in membrane-bound states. Considering the locations of A13 in state II/II (that is, 7.6??/>10?? down from head group phosphates), the neighbouring D14 side chain should also be among the GDC-0879 hydrophobic alkyl chains, and thus may already be protonated at pH 6.4C7.4. The linker region between A13 and A27 is long and hydrophobic, with the sequence WLFTTPLLLLDL between residues 15 and 26. During state GDC-0879 II to state II transition, to have such a dramatic pulling effect on A27 (from >10?? outside the membrane in state II to 6.40.2?? in state II), the D25 side chain may also become kinetically protonatable at pH 6.4. We do not know whether A27 is located 6.4?? above or below the phosphate groups, and therefore, both models are presented in Fig. 7. Once D25 is protonated, the LALLV linker between residues 26 and 30 may in turn pull D31, D33 and E34 of the C terminus into membrane environments for protonation, ultimately reaching the equilibrium ratio of 30% inserted (that is, state III) : 70% embedded (that is, state II) at pH 6.4. In this proposed model, the protonation of D25 is the master switch that controls insertion, which is consistent with the fact that small modifications in D25 side chain can significantly alter the pH50 of insertion27,39. The interlacing of polar but neutralizable Asp residues and hydrophobic sinker stretches (that is, WLFTTPLLLL between D14 and D25 and LALLV between D25 and D31) may allow pHLIP to respond to slight change in acidity with impressive cooperativity. Future NMR characterization will be GDC-0879 performed to study the pH-dependent protonation states of these key Asp residues. Last, these data also raise the possibility that pHLIP may respond to slight acidity in cellular GDC-0879 and experiments through the state II to state II transition, with the latter intermediate state II GDC-0879 supporting facile insertion by anchoring the C-terminal region surrounding D25 more deeply in the membrane. Methods Peptide synthesis and purification All the isotope-labelled pHLIP (NH2-GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-CO2H, with labelled sites shown in bold) were synthesized manually using the Rabbit Polyclonal to APLF. routine 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group chemistry with preloaded Fmoc-Thr(for 1?h at 4?C. The gel-like wet pellet was packed into a solid-state NMR rotor using an Eppendorf centrifuge. The 280?nm absorbance of the supernatant was measured to ensure that >90% of pHLIP peptides are with the POPC liposomes. Tryptophan fluorescence assays For Trp fluorescence experiments, the pHLIP/POPC solutions prepared as described above were adjusted to a series of samples with 20 different pH values using concentrated buffer solutions (containing 50?mM of sodium phosphate and 50?mM of sodium acetate). The final pH values ranged from 8.12 to 4.00. The actual pH values after the adjustment was determined right before the fluorescence measurements. Each solution sample at different pH values had an associated control sample that was prepared from the liposomes without pHLIP. All fluorescence spectra were recorded on a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer Inc., Waltham, MA). The samples were excited at 285?nm with 10?nm slit width and the emission spectra were collected from 301 to 400?nm with 2.5?nm slit width and 300?nm?min?1 scanning rate. For each sample, the associated background signal was subtracted from the raw data. The maximum peak wavelength was determined using the FL WinLab software associated with the spectrometer. CD spectroscopy A lyophilized sample of pHLIP was dissolved in aqueous sodium phosphate (NaPi) buffer (1?mM, pH 8.2) to create a 10?M.