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

Abstract

Large, hydrophobic residues (isoleucine, leucine, and valine) dominate sequences of transmembrane (TM) helices in membrane proteins (total ∼34%), but their relative roles in mediating the biologically relevant protein–lipid and protein–protein interactions have not been systematically evaluated. Here we have synthesized Leu‐containing Lys‐tagged hydrophobic peptides of identical composition, where sequences have been designed with their Leu residues either scrambled (sequence KKKLAASALAAAWLAALALSAAKKK); clustered (KKKAAASAALLLWLLAAAASAAKKK); or “lipopathic” (all Leu on one helical face) (KKKAAASLAALLWALLAAASAAKKK). These peptides were compared by several biophysical/biochemical techniques to the corresponding set of peptides where the Leu residues are replaced by the isosteric Ile residues. Circular dichroism spectra showed that all peptides were helical in POPC liposomes, as confirmed by blue shifts in Trp fluorescence spectra, notably with the Ile‐lipopathic peptide displaying increased Trp burial versus its Leu counterpart. Quenching experiments with a dibromo‐PC lipid indicated deeper membrane penetration of the Ile versus the Leu lipopathic peptide—a result supported by protease degradation assays where Ile peptides reconstituted into lipid bilayers were significantly more protected from the protease than the Leu peptides. Assessment of Trp blue shifts in the presence of lipid bilayers of varied lipid packing indicated that Leu/Ile peptide interactions are dependent on lipid composition. The overall results suggest that two main interactions tend to dominate Leu and Ile interactions within the membrane: (1) hydrophobic interactions between amino acid side chains and the surrounding lipid; and (2) degree of disruption of lipid–lipid packing. This “battle of giants” likely underlies the specific role(s) that Leu and Ile will play in the folding of a given membrane protein.

The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs

“…Another possibility is that Leu provides better interactions with lipids than Ile and is therefore responsible for hydropathy tuning. Due to the difference in branching position, surface‐exposed Leu residues possess higher hydrophobic side chain densities remote to the helix backbone thereby providing more surface for forming interactions with lipid tails compared to Ile 3 . Thus, Leu provides increased protein‐lipid interactions, which likely becomes advantageous for proteins within less densely packed, fluid membranes, as has been shown by Deber and Stone using a peptide model system 3 .…”

“…Due to the difference in branching position, surface‐exposed Leu residues possess higher hydrophobic side chain densities remote to the helix backbone thereby providing more surface for forming interactions with lipid tails compared to Ile 3 . Thus, Leu provides increased protein‐lipid interactions, which likely becomes advantageous for proteins within less densely packed, fluid membranes, as has been shown by Deber and Stone using a peptide model system 3 . Differences in how the two amino acids interact with lipids suggest that membrane properties (such as lipid composition) might influence the degree to which Leu or Ile (or potentially other amino acids) tune TMD hydropathy.…”

“…Most well‐known is that Ile has a lower propensity to be within α‐helices due to steric clashes caused by the β‐branching 1,2 . In addition, another study showed differences in how the two amino acids interact with lipids 3 . However, little is known if there are additional general trends that distinguish the two amino acids within proteins.…”

Molecular de-extinction of antibiotics enabled by deep learning

“…S27 ), AEPs have a high abundance of uncharged polar residues 18 . Leucine and isoleucine, in particular, are structurally important: the stiffness of these bulky branched residues limits the internal flexibility of the peptide, whereas other aliphatic residues favor specific foldamers during the folding process 22 . The difference between the amino acid composition of known AMPs and that of AEPs and MEPs results in significantly different physicochemical features ( Fig.…”

Predicting Cell-Penetrating Peptides: Building and Interpreting Random Forest based prediction Models

“…It was also observed that transmembrane helices are enriched in Leu residues and oligoleucines can insert themselves into membranes (Gurezka et al, 1999;Deber and Stone, 2019). It will be interesting to probe further the role of Leu in CPPs.…”