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

Abstract

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...

Cooperative Non-bonded Forces Control Membrane Binding of the pH-Low Insertion Peptide pHLIP

“…3A). The fact that wt-pHLIP and pHLIP-1 undergo folding into helical turns in different segments of the peptide is consistent with proposed models of the reversible state II to state III transition (14,39,50,51). In wt-pHLIP, folding mainly occurs from I7-Y12 and D14-T18 but also in the sinker stretch from L21-V30.…”

“…In this study we have computationally modeled state II of wt-pHLIP and the variant, pHLIP-1. This is the first computational study of its kind, as previous MD studies have focused on insertion of α-helical pHLIP (38,39), conformational sampling of gold nanoparticle-pHLIP conjugates (40), and pHLIP in solution (14,19). In contrast, the field of antimicrobial peptides and cell-penetrating peptides has been extensively studied by MD simulations (41)(42)(43).…”

“…Although they observed two populations of conformations at pH 6.4, the C-terminus (i.e., the "sinker stretch") was helical for both, indicating that helix formation most likely initially occurs around the D25 protonation switch. In their latest study, 13 C solid-state NMR was used to determine that the C-terminal aspartic acid residues undergo titration first (i.e., highest pK a 's), leading An, Qiang, and coworkers to postulate that the solvent-exposed residues on the C-terminus underwent protonation under acidic conditions more quickly than residues in the middle of the peptide (D14 and D25) (14). By combining the insights gained from our simulations with these experimental observations of pHLIP, we can begin to formulate a general mechanism for binding of pHLIP in state II.…”

“…In pHLIP-1, folding is almost completely conserved from Y12-T18 and can extend from L22-V30. Based on solid-state NMR and fluorescence spectroscopy studies, the C-terminal half of the transmembrane segment of pHLIP is most sensitive to changes in pH ( (14,51) and can form a peripheral helix prior to insertion (50). In addition, recent constant-pH MD studies that were initiated in state III showed that at pH 7, the N-terminal half of the transmembrane segment (i.e., including D14) diffuses towards the surface of the bilayer but remains in an α-helical fold (39).…”

“…The C-terminal residues that were isotopically labeled (L21, L22, L26, and A27) displayed a high degree of dynamics, leading to their hypothesis that the polar C-terminus of pHLIP (D31, D33, and E34) plays a major role in preventing partitioning of that end of pHLIP into the bilayer surface. A subsequent follow-up study from An and Qiang showed that the acidic residues in the C-terminus of pHLIP not only affect the ability for pHLIP to partition into the bilayer surface, but they are also the first residues to be titrated upon acidification of the surrounding environment (14). What was unclear was the specific relationship between binding of pHLIP to the bilayer surface and titration of acidic residues -is titration facilitated by a change in hydrophobicity of the surrounding environment through burial in the headgroup region, or does exposure to solvent, as most likely with the C-terminus of pHLIP, allow for faster transitions from the acidic to the neutral state?…”