The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization

To explore the influence of amino acid composition on the behavior of membr ane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleu cyl (pLeu) helices containing a single polar/ionizabIe residue within their hydrophobic core. To evaluate the location of the helices within the membr ane by fluorescence, each contained a Trp residue at the center of the sequ ence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membr ane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp r esidue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. Ln this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide- labeled lipids showed it located at the bilayer center. A state in which Tr p fluorescence red-shifted by several nanometers (named the B state) was ob served above pH 10-11. B state formation appears to result from deprotonati on of the flanking Lys residues. Despite the red shift in Trp emission, flu orescence quenching showed that in the B state the Trp at most is only slig htly shallower than in the N state, suggesting the B state also is a transm embrane or near-transmembrane structure. The B state is characterized by in creased helix oligomerization, as shown by the dependence of Trp lambda(max ) on the concentration of the peptide within the bilayer at high pH. The pL eu peptide with a Asp residue in the core underwent a pH-dependent transiti on at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location tha n the other peptides. This state (named the S state) did not exhibit a conc entration-dependent Trp lambda(max). We attribute S state behavior to the f ormation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontra nsmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ioni zable residues in the core and regions flanking the helix significantly mod ulate the stability of transmembrane insertion and/or helix-helix associati on.