Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Proc Natl Acad Sci U S A. 2016 Sep 13;113(37):10340-5. doi: 10.1073/pnas.1605888113. Epub 2016 Aug 25.


The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (

Keywords: Bellman–Ford search; membrane insertion; positive-inside rule; topology prediction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence / genetics
  • Amino Acids / chemistry
  • Cell Membrane / chemistry*
  • Cell Membrane / genetics
  • Cytoplasm / chemistry
  • Cytoplasm / genetics
  • Energy Metabolism / genetics
  • Hydrophobic and Hydrophilic Interactions*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Protein Conformation*


  • Amino Acids
  • Membrane Proteins