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.

The pH-low insertion peptide (pHLIP) inserts into membranes and forms a transmembrane (TM) α-helix in response to slight acidity, and has shown great potential for cancer diagnosis and treatment. As a lead, pHLIP is challenging to optimize because the mechanism of its pH-dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change, the particular role played by each of them in the protonation-driven insertion process is not clear. The precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid-state NMR spectroscopy is used to address these central questions. Tracing backbone conformations revealed that the TM helix spans from A10 to D33 with a break at T19 to P20. Residue-specific pKa values of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively, and define the sequence of protonations which lead to insertion. Furthermore, possible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.

Interactions between the β amyloid (Aβ) peptides and cellular membranes have severe consequences such as neuronal cell disruption and therefore may play important roles in Alzheimer's disease. Understanding the structural basis behind such interactions, however, is hindered by the complexity of the Aβ-membrane systems. In particular, because the Aβ peptides are partially incorporated in the membrane bilayer after enzymatic cleavage, there are multiple possibilities in terms of the initial proximity between the peptides and membranes. Structural studies using in vitro model systems with either externally added or preincorporated Aβ in membrane bilayers resulted in distinct evolution pathways. Previous work has shown that the externally added Aβ formed long and mature filaments, while preincorporated Aβ generated short and curvy fibrils. In this study, we perform detailed characterizations on the structural evolution and membrane interaction for these two pathways, using a combination of solid-state nuclear magnetic resonance spectroscopy and other techniques. For the externally added Aβ, we determined the residue-specific structural evolution during the fibrillation process. While the entire fibrillation process for the externally added Aβ was slow, the preincorporated Aβ generated Aβ-lipid complexes rapidly. Specific interactions between the lipids and peptides were observed, suggesting the colocalization of lipids and peptides within the complex. Formation of such a complex induced molecular-level changes in the lipid bilayer, which may serve as a possible mechanism of membrane disruption.

The pH-lowi nsertion peptide (pHLIP) inserts into membranes and forms at ransmembrane (TM) a-helix in response to slight acidity,a nd has shown great potential for cancer diagnosis and treatment. As alead, pHLIP is challenging to optimize because the mechanism of its pH-dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change,t he particular role played by eacho ft hem in the protonation-driven insertion process is not clear.T he precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid-state NMR spectroscopyi su sed to address these central questions.T racing backbone conformations revealed that the TM helix spans from A10 to D33 with abreak at T19 to P20. Residue-specific pK a values of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively,a nd define the sequence of protonations which lead to insertion. Furthermore,p ossible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.The 36-residue (GGEQNPIYWARYA DWLFTTPLLLL-DLALLVDADEGT) pH-low insertion peptide (pHLIP), which is extracted from the bacteriorhodopsin helix C, can sense microenvironmental pH change and inserts into lipid bilayers as atransmembrane (TM) a-helix below acritical pH value. [1][2][3][4][5][6] Thepeptide is largely unfolded in aqueous solution (state I) and on the membrane surface (state II), and forms the folded state III upon insertion. [4] Biomedical applications of pHLIP (and its analogues) have attracted increasing attentions because of their controllable responses to slight acidity in the pH range of 6-7 found in various diseased states. They have been utilized as imaging tools and diagnostic agents for cancer, inflammation, and ischaemic myocardium. [1,4,5,[7][8][9][10] Furthermore,p HLIP technology constitutes an ew,p H-responsive,c ross-membrane cytoplasmic cargo delivery platform. 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. [11][12][13][14][15][16][17][18][19][20] To extend the biomedical applications of pHLIP to broader systems with higher efficiency,a ppropriate tuning of its primary sequence is necessary, [21,22] and to do so requires an in-depth and detailed understanding of its pH-regulated membrane insertion process,which is currently not available.Them acroscopic, apparent pH 50 ,d efined as the pH at which 50 %o ft he pHLIP molecules are inserted (also referred to as pK or pK a of insertion by others), has been estimated to be about 6.1-6.2 by following fluorescence changes of Wresidues at positions 9and 15. [1,4] However,this assay is indirect and it is not clear which protonations of the D/E side chains within the pHLIP sequence correlate with aWfluorescence blue-shift and an increase of fluorescence intensity.The original hypothesis of how pH regulates pHLIP insertion rest...

No abstract