Residue-specific structures and membrane locations of pH-low insertion peptide by solid-state nuclear magnetic resonance

Abstract

The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II′ that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

Protonation‐Driven Membrane Insertion of a pH‐Low Insertion Peptide

“…[4,29,30] Figures 3A-D show tryptophan quenching by acrylamide or 10-DN,thus indicating the exposure of Wside chains to water or hydrophobic membrane interior,respectively. [25] Therefore,weexpect W9 to be near the headgroup phosphate level (or slightly above) and W15 below the head-group/ alkyl chain interface.S uch membrane positions may lead to preferential quenching of the more exposed W9 by the aqueous phase quencher acrylamide,a nd the more deeply buried W15 by the lipid phase quencher 10-DN.T he acrylamide/W9 pairing is also consistent with earlier published fluorescence decomposition study showing only one of the two Wr esidues is accessible to acrylamide in state II. We learned previously from ssNMR data that at both pH 7.4 (state II) and 5.3 (state III): a) the b-carbon atom of A10 and A13 are located about 5.7 and 7.6-7.8 ,r espectively,a way from head-group 31 Pn uclei;a nd b) A13 has contacts with lipid alkyl chains.…”

“…They have been utilized as imaging tools and diagnostic agents for cancer, inflammation, and ischaemic myocardium. [25] Herein we fully characterize the secondary structures of pHLIP in the inserted state III (pH 5.3, pHLIP/ POPC ratio:1 :75). Avariety of molecules,i ncluding fluorescent dyes,cytotoxins,chemotherapy drugs,and biomolecules, such as peptide nucleic acids (PNA), have been delivered into cells using pHLIP.…”

“…[25] Herein we fully characterize the secondary structures of pHLIP in the inserted state III (pH 5.3, pHLIP/ POPC ratio:1 :75). [25] Herein we fully characterize the secondary structures of pHLIP in the inserted state III (pH 5.3, pHLIP/ POPC ratio:1 :75).…”

“…[25] Herein we fully characterize the secondary structures of pHLIP in the inserted state III (pH 5.3, pHLIP/ POPC ratio:1 :75). [26] 3) Taken together with previous observations that A10 and A13 are located about 5.7 and 7.8 away from 31 P( assumed to be head groups of outer leaflet lipids) in state III, [25] it is very likely that the Cterminus of pHLIP is located in the phosphate head-group region of the inner leaflet of the bilayer because the 31 P-to-31 P thickness is about 38 for POPC and 38-43 for plasma membranes in mammalian cells.I no ther words,p HLIP is barely long enough to be truly transmembrane. There are several important conclusions that can be drawn from these NMR data:1)The backbone a-helical conformation starts from A10 and ends at D33 and gives has a3 6 span.…”

“…Protonation of aDside-chain carboxylate induces asignificant Cg upfield shift which can be monitored by ssNMR spectroscopy. Since the C-terminal half of pHLIP (L21 to T36) contains mainly hydrophobic and D/E residues,p rotonations of D31 and D33 residues would dramatically reduce the hydrophilicity of the C-terminal region, thus allowing pHLIP to sink deeper into the membrane (pH 7.4 state II to pH 6.4 state II' transition), [25] and in turn lead to subsequent D25 and D14 protonations and ultimately membrane insertion. TheDQF was applied to remove the natural abundance carbonyl signal from lipids.…”