Probing inner membrane protein topology by proteolysis running head: Protease accessibility

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