Abstract
The de novo design of integral membrane proteins remains a major challenge in protein chemistry. Here, we describe the bottom-up design of a genetically-encoded synthetic membrane protein comprising only four amino acids (L, S, G and W) in the transmembrane domains. This artificial sequence, which we call REAMP for recombinantly expressed artificial membrane protein, is a single chain of 133 residues arranged into four antiparallel membrane-spanning α-helices. REAMP was overexpressed in Escherichia coli and localized to the cytoplasmic membrane with the intended transmembrane topology. Recombinant REAMP could be extracted from the cell membrane in detergent micelles and was robust and stable in vitro, containing helical secondary structure consistent with the original design. Engineered mono- and bis-histidine residues in the membrane domain of REAMP were able to coordinate heme in vitro, in a manner reminiscent of natural b-type cytochromes. This binding shifted the electrochemical potential of the cofactor, producing a synthetic hemoprotein capable of nascent redox catalysis. These results show that a highly reduced set of amino acids is sufficient to mimic some key properties of natural proteins, and that cellular biosynthesis is a viable route for the production of minimal de novo membrane sequences.
Highlights
Integral membrane proteins play vital roles in biological systems including solute transport, signal transduction, and energy generation
Because our intention was to design a four-helix bundle, members of this family were of interest since the SMR protomer contains four contiguous and hydrophobic TM helices connected by short extramembrane loops
The results presented here show that the minimal de novo sequence REAMP is expressible, can be successfully processed by the cell, and is stable after purification. The implications of this are discussed below. It is well-attested that fusion proteins can support the expression of synthetic transmembrane segments, it is less clear that this extends to standalone de novo sequences and examples are relatively scarce
Summary
Integral membrane proteins play vital roles in biological systems including solute transport, signal transduction, and energy generation. The most powerful insights are likely to come from simple minimalist sequences that offer a neutral design background This is borne out by the extensive use of model transmembrane helices with limited sequence diversity to investigate membrane insertion[6,7,8,9,10], topology[11,12,13], folding[14,15,16,17,18] and assembly[19,20,21]. We present a new minimal de novo protein that can be successfully expressed into the cytoplasmic membrane of E. coli and subsequently purified for further study. Our findings establish REAMP as a novel biocompatible scaffold that can imitate some features of natural membrane proteins
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