Probing Inner Membrane Protein Topology by Proteolysis

Methods Mol Biol. 2017:1615:97-103. doi: 10.1007/978-1-4939-7033-9_8.


Inner membrane proteins are inserted into the membrane via α-helices. These helices do not only constitute membrane anchors but may mediate specific interactions with membrane protein partners or participate in energetic processes. The number, location, and orientation of these helices is referred to as topology. Bitopic membrane proteins that consist of a single membrane-embedded domain connecting two soluble domains are distinguished from polytopic ones that consist of multiple membrane-spanning helices connected by extramembrane domains. Defining inner membrane protein topology could be achieved by different methods. Here we describe a protease accessibility assay that makes it possible to define topology based on digestion profiles.

Keywords: Bitopic; Carboxypeptidase Y; Inner membrane; Insertion; Membrane protein; Polytopic; Protease; Proteinase K; Proteolysis; Topology; Transmembrane segment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Protein Structure, Secondary*
  • Proteolysis
  • Spheroplasts / isolation & purification


  • Bacterial Proteins
  • Membrane Proteins
  • Endopeptidases