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 membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In 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 Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition 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 than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation 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 nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.
Department of Biochemistry and Cell Biology and Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
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London
D000596:Amino Acids / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry; D010713:Phosphatidylcholines; D017433:Protein Structure, Secondary
D000596:Amino Acids; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D010713:Phosphatidylcholines; C027819:polyleucine; C017251:1,2-oleoylphosphatidylcholine; D008239:Lysine