The ability of the pH-Low Insertion Peptide (pHLIP) to insert into lipid membranes in a transbilayer conformation makes it an important tool for targeting acidic diseased tissues. pHLIP can also serve as a model template for thermodynamic studies of membrane insertion. We use intrinsic fluorescence and circular dichroism spectroscopy to examine the effect of replacing pHLIP's central proline on the pH-triggered lipid-dependent conformational switching of the peptide. We find that the P20G variant (pHLIP-P20G) has a higher helical propensity than the native pHLIP (pHLIP-WT), in both water:organic solvent mixtures and in the presence of lipid bilayers. Spectral shifts of tryptophan fluorescence reveal that with both pHLIP-WT and pHLIP-P20G, the deeply penetrating interfacial form (traditionally called State II) is populated only in pure phosphocholine bilayers. The presence of either anionic lipids or phosphatidylethanolamine leads to a much shallower penetration of the peptide (referred to here as State II, for "shallow"). This novel state can be differentiated from soluble state by a reduction in accessibility of tryptophans to acrylamide and by FRET to vesicles doped with Dansyl-PE, but not by a spectral shift in fluorescence emission. FRET experiments indicate free energies for interfacial partitioning range from 6.2 to 6.8kcal/mol and are marginally more favorable for pHLIP-P20G. The effective pKa for the insertion of both peptides depends on the lipid composition, but is always higher for pHLIP-P20G than for pHLIP-WT by approximately one pH unit, which corresponds to a difference of 1.3kcal/mol in free energy of protonation favoring insertion of pHLIP-P20G.
Overexpression and deregulation of the epidermal growth factor receptor (EGFR) are implicated in multiple human cancers and therefore are a focus for the development of therapeutics. Current strategies aimed at inhibiting EGFR activity include monoclonal antibodies and tyrosine kinase inhibitors. However, activating mutations severely limit the efficacy of these therapeutics. There is thus a growing need for novel methods to inhibit EGFR. One promising approach involves blocking the association of the cytoplasmic juxtamembrane (JM) domain of EGFR, which has been shown to be essential for receptor dimerization and kinase function. Here, we aim to improve the selectivity and efficacy of an EGFR JM peptide mimic by utilizing the pH(low) insertion peptide (pHLIP), a unique molecule that can selectively target cancer cells solely based on their extracellular acidity. This delivery strategy potentially allows for more selective targeting to tumors than current methods and for anchoring the peptide mimic to the cytoplasmic leaflet of the plasma membrane, increasing its local concentration and thus efficacy. We show that the conjugated construct is capable of inhibiting EGFR phosphorylation and downstream signaling and of inducing concentration- and pH-dependent toxicity in cervical cancer cells. We envision that this approach could be expanded to the modulation of other single-span membrane receptors whose activity is mediated by JM domains.
The interaction of membrane proteins with and within lipid membranes is vital to a plethora of cellular processes from the control of intracellular signaling by peripheral membrane proteins, to the activity of antimicrobial peptides and integral membrane proteins. Thus, quantifying the mechanism by which proteins interact with and within lipid membranes is essential to understanding their biological functions. However, many protein-lipid and protein-protein interactions remain poorly understood due to the difficulty in studying processes that occur at the membrane surface. There is thus a clear need for a sensitive, versatile, non-perturbing and physiologicallyrelevant biophysical method capable of measuring association constant and stability free energy of membrane proteins in a wide range of conditions and matrices. Backscattering interferometry (BSI) is an analytical technique that can monitor and quantify molecular interactions through the detection of small changes in refractive index (RI) induced by molecular interaction. BSI offers many advantages over current techniques including: no need for label or surface immobilization, small sample sizes (1-2 mL), low concentrations (pM to mM), remarkable sensitivity, broad dynamic range for dissociation constant, and low cost. Here, the potential application of BSI in the field of membrane proteins is illustrated through three case studies: (1) Association of small peptides with lipid vesicles, (2) transmembrane helix dimerization, and (3) unfolding of integral membrane proteins. Our preliminary results suggest that BSI is amenable to the study of such systems.
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