The de novo design of a biocompatible and functional integral membrane protein using minimal sequence complexity

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.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

“…S11, Supplementary Table 3). The potentials measured here are very similar to those previously obtained for REAMP bis-His variants that can complex heme 24 , implying that the immediate heme environment is consistent between the two designs.…”

“…Either decahistidine or triple StrepII-tag sequences were placed at the Protein purification. Protein purification from E. coli was as previously described 24 with no modifications. The process is outlined in Fig.…”

“…Heme (as hemin) or zinc protoporphyrin IX were introduced and samples incubated at 25 °C for equilibrium measurements. Redox potentiometry was performed in the presence of mediators as before 24 . For kinetic analysis, heme and protein were mixed at 1 μM each reactant in a stopped-flow instrument in absorption mode.…”

“…We previously found that REAMP H/H was marginally active as a heme peroxidase 24 , and so wondered if the dynamical changes observed in REAMP2.0 would affect this activity. Peroxidase assays confirmed that REAMP2.0 and its histidine variants were substantially more active than analogous REAMP complexes ( Fig.…”

Towards functional de novo designed proteins

“…Following a tradition established by Lear et al [42], Lalaurie et al employ minimal design to deliver a de novo membrane protein [43]. By analysing a small subset of natural membrane proteins, the authors develop a low-complexity leucine-rich sequence.…”