Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1860, Issue 2, Pages 534-543Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2017.11.006
Keywords
pH-dependent membrane insertion; Tryptophan fluorescence; Thermodynamics; Conformational switching; Transmembrane helix
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Funding
- National Institutes of Health [P30 GM110761, R21CA181868]
- Lehigh University
- NATIONAL CANCER INSTITUTE [R21CA181868] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P30GM110761] Funding Source: NIH RePORTER
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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 Il(s), 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 6.8 kcal/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 1.3 kcal/mol in free energy of protonation favoring insertion of pHLIP-P20G.
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