pH-dependent thermodynamic intermediates of pHLIP membrane insertion determined by solid-state NMR spectroscopy

Abstract

The applications of the pH low insertion peptide (pHLIP) in cancer diagnosis and cross-membrane cargo delivery have drawn increasing attention in the past decade. With its origin as the transmembrane (TM) helix C of bacteriorhodopsin, pHLIP is also an important model for understanding how pH can affect the folding and topogenesis of a TM α-helix. Protonations of multiple D/E residues transform pHLIP from an unstructured coil at membrane surface (known as state II, at pH ≥ 7) to a TM α-helix (state III, pH ≤ 5.3). While these initial and end states of pHLIP insertion have been firmly established, what happens at the intervening pH values is less clear. However, the intervening pH range is most relevant to pHLIP−cell interactions in the acidic extracellular tumor environment (and in the endosomes within cells). Here, using advanced solid-state NMR spectroscopy with palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine unilamellar vesicles as the model membrane, we systematically examined the state of pHLIP−membrane interactions (in terms of the membrane locations of D/E residues, as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate states distinct from the initial and end states were discovered to exist at each of the intervening pH examined. They support a multistage model of pHLIP insertion in which the D/E titrations occur in a defined sequence at distinct intermediate pH values. This multistage model has important ramifications in pHLIP applications.

Kinetics of pHLIP peptide insertion into and exit from a membrane

“…The transition correlates with our recent constant-pH molecular dynamics (MD) calculations, which suggest a high flexibility of the N-terminal flanking sequence of pHLIP (4). At different pH values in the range of 8.5-6.5, pHLIP can adopt various conformational states at a bilayer surface, as was noticed previously (6,8). These conformational changes are not associated with helix formation.…”

“…Recently, a multistage model of WT-pHLIP insertion with distinct equilibrium thermodynamic intermediates has been proposed (8). While it is interesting to think there is a linear progression of states, the fact is that an ensemble of all peptide states exists at each pH, so it remains challenging to define a unique succession.…”

“…Because their membrane insertion from a water-soluble state is triggered by pH changes, a rich opportunity is created for chemical, kinetic, and computational studies, as shown by the expanding literature from a growing number of laboratories and ongoing efforts to use them as medically useful acidity sensors in vivo. Currently, pHLIPs are being used in studies of membrane-associated folding and unfolding as a model system (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) and in a variety of biomedical applications for targeted delivery of imaging and therapeutic agents (11)(12)(13)(14). In this paper, we position Tryptophans (Trp) as sensors in the flanking and central regions of a pHLIP and exploit kinetic analysis to study the pathways of membrane entry and exit.…”

Targeting Acidic Diseased Tissues by pH-Triggered Membrane-Associated Peptide Folding

“…At intermediate pHs a more distributed mixture of states is present. Therefore, any assumption of a sequential progression of intermediates from the membrane-adsorbed to the membrane-inserted state (Otieno et al, 2018) is a simplified view, which does not reflect the complexity of thermodynamic ensembles in real physical systems. • The shift of the equilibrium from one distribution of states to another is triggered by pH changes in the environment near the lipid bilayer: a drop of pH induces protonation of Asp/Glu residues and the C-terminus.…”

Ions Modulate Key Interactions between pHLIP and Lipid Membranes

“…The majority of biophysical studies on pHLIP have focused on modifications to the peptide and the resulting changes to function based on interactions with vesicles of the model lipid 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) (4,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). However, the role of environmental factors and their effect on the function of pHLIP have recently garnered attention.…”