Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

Abstract

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.Cells are defined by a thin membrane that separates the inside of the cell from the outside. The core of this membrane is hydrophobic, meaning that it repels water. Many signals and nutrients cannot pass through the membrane itself, but can pass through the proteins that span the membrane. Membrane proteins are therefore essential for living cells; yet even after decades of research, it remains unclear how proteins interact with the membrane and which features determine a protein’s stability in a biological membrane.Since the early 1980s it was known that the bacterium E. coli could grow on a common antibiotic called ampicillin if it had enough of an antibiotic-degrading enzyme called β-lactamase anchored into its inner membrane. Now, Elazar et al. have used this enzyme to obtain detailed information on the interactions between a biological membrane and a membrane protein. First, hundreds of different mutations were introduced into the gene that encodes the enzyme to generate a population of bacteria that each had a slightly different membrane anchor. The mutant bacteria were then grown in the presence of the antibiotic, meaning that those mutants with a more stable membrane anchor were more likely to survive and grow than those with less stable anchors.Elazar et al. then collected all the surviving bacteria, sequenced their DNA and measured how common the different mutations were in the final population. This approach was less labor-intensive and more accurate than traditional methods for monitoring membrane-anchored proteins, and the resulting large dataset was used to uncover which features affect a protein’s stability in a membrane. These results also showed that a biological membrane’s core is considerably more hydrophobic than was previously thought.In addition to being hydrophobic, biological membranes have more negative charge in the side that faces into the cell. This means that membrane proteins with a positive charge in this region will be more stable, and Elazar et al. were able to use their new system to measure this effect for the first time.Finally, membrane proteins do not only span the membrane; they also bind with other membrane proteins in order to carry out their roles. Elazar et al. used their system to look at the surfaces of human membrane proteins that interact with one another, and build a detailed map of the interaction surfaces, from which they derived accurate models of the membrane proteins.Overall, these new findings could now be used to model the three-dimensional structures of membrane proteins and improve their stability. This in turn may help efforts to develop these proteins into more robust experimental tools and in the search for drugs that target membrane proteins.