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.

Biological insertion of computationally designed short transmembrane segments

“…More recently, TM disposition of poly-Leu sequences were analyzed using synthetic peptides and oriented phospholipid bilayers, in vitro insertion into microsomal membranes and molecular dynamics simulations 5 . Sequences with either blocks of or dispersed hydrophobic residues have also been analyzed using similar methods 6 . The picture that emerges from these studies is that lipid bilayers adapt to TM helices as short as 10–12 leucines.…”

Cell Surface and Membrane Engineering: Emerging Technologies and Applications

“…The increasing success and robustness of this technique may soon make the expression of membrane proteins in NLPs a common and routine laboratory technique that becomes standard for the general study and exploitation of membrane proteins. As a caveat, many proteins do not spontaneously insert into lipid bilayers and therefore are not immediately amenable for NLP incorporation; however as the basic requirements for protein insertion into membranes become unraveled [ 61 , 62 , 63 ] it is anticipated that peptide sequences can be rationally designed to widely facilitate NLP production for an increased repertoire of proteins.…”

Relative role(s) of leucine versus isoleucine in the folding of membrane proteins

“…Previous work from our lab has focused on the use of designed hydrophobic peptides to analyze the dependence of hydrophobic patterning in lipid environments . In those studies, we predominantly used Leu to represent typical hydrophobic amino acids within TM domains.…”