title: Hydrophobic blocks facilitate lipid compatibility and translocon recognition of transmembrane protein sequences.
abstract: Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.
authors: Division of Molecular Structure & Function, Research Institute, Hospital for Sick Children , Toronto M5G 0A4, Ontario, Canada.
authors: Tracy A
authors: TA
authors: Stone
authors: Nina
authors: N
authors: Schiller
authors: Gunnar
authors: G
authors: von Heijne
authors: Charles M
authors: CM
authors: Deber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004285:Dogs; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D012694:Serine; D007930:Leucine
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