Abstract
The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design.
Highlights
The great majority of helical membrane proteins are inserted co-translationally into the endoplasmic reticulum (ER) membrane through a continuous ribosome-translocon channel
It is well accepted that thermodynamically favorable partitioning of TM helices from the translocon into the more hydrophobic environment is important at the insertion stage[2,3], the limits for the insertion of TM helices with naturally occurring amino acid distributions has not been systematically explored
While the studies cited above are all based on model hydrophobic sequences composed of only a few different kinds of amino acids, TM helices of amino acid composition that more closely matches natural TM helices need to be studied to lay a foundation for more advanced TM helix designs
Summary
We swapped the histidine residue with its neighboring valine residue (L-K23 H3V/V4H, Table 1) generating an (i, i + 2) periodicity for the His-Glu pair and likely precluding intrahelical pairing by orienting the two side-chains toward opposite faces of the helix This mutant resulted in a slightly increased predicted pi value but consistently diminished experimental insertion efficiency. The combined effect of these data improved the correlation between the experimental and prediction values for the mutant sequence (Fig. 5, arrow pointed dot), which has the same amino acid composition as the L-K23 sequence These results support the idea that intra-helical salt-bridge formation between residues located on the same face of a TM helix (i, i + 3) may reduce the free energy of membrane partitioning[9,10], whereas the presence of His and Glu on opposite faces of the helix (i, i + 2) is unfavorable and lowers the ER translocon membrane insertion efficiency. In the case of A6P/P10A mutant (− 6 position) the presence of the proline closer to the interface would locate the unsatisfied carbonyl group in a less hydrophobic environment[13,14], probably reducing the free energy of membrane partitioning
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