TY - JOUR
T1 - Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane
AU - Assaf, Elazar
AU - Weinstein, Jonathan
AU - Biran, Ido
AU - Fridman, Yearit
AU - Bibi, Eitan
AU - Fleishman, Sarel-Jacob
N1 - We thank Olga Khersonsky, Dror Baran, Adina Weinberger, Jens Meiler, and Zohar Mukamel for advice, and Gunnar von Heijne, Nir Ben-Tal, Ingemar Andre, Julia Koehler-Leman, and Dan Tawfik for critical reading. Ilan Samish and William DeGrado provided advice for analyzing knowledge-based insertion energies. The research was supported by the Minerva Foundation with funding from the Federal German Ministry for Education and Research, a European Research Council’s Starter’s Grant, an individual grant from the Israel Science Foundation (ISF), the ISF’s Center for Research Excellence in Structural Cell Biology, career development awards from the Human Frontier Science Program and the Marie Curie Reintegration Grant, an Alon Fellowship, and a charitable donation from Sam Switzer and family. Funding - Minerva Foundation: Assaf Elazar Jonathan Weinstein Yearit Fridman Sarel Jacob Fleishman European Research Council: Assaf Elazar Jonathan Weinstein Yearit Fridman Sarel Jacob Fleishman The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Contribution: AE, Conducted the insertion and association deep sequencing experiments, Conducted the western blot experiments, Wrote software to analyze the deep-sequencing data, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. JW, Analyzed structure-based potentials, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. IB, Conducted the western blot experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. YF, Conception and design, Acquisition of data. EB, Conception and design, Analysis and interpretation of data, Drafting or revising the article. SJF, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.
PY - 2016/1/29
Y1 - 2016/1/29
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84962576764&partnerID=8YFLogxK
U2 - https://doi.org/10.7554/eLife.12125
DO - https://doi.org/10.7554/eLife.12125
M3 - مقالة
SN - 2050-084X
VL - 5
JO - eLife
JF - eLife
IS - JANUARY2016
M1 - e12125
ER -