title: Aspartate embedding depth affects pHLIP's insertion pKa.
abstract: We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520, United States.
authors.forename: Justin
authors.initials: J
authors.lastname: Fendos
authors.forename: Francisco N
authors.initials: FN
authors.lastname: Barrera
authors.forename: Donald M
authors.initials: DM
authors.lastname: Engelman
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D002942:Circular Dichroism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D008565:Membrane Proteins; C533201:pHLIP protein; D001224:Aspartic Acid