Probing Inner Membrane Protein Topology by Proteolysis
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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.
Key wordsMembrane protein Inner membrane Insertion Topology Transmembrane segment Bitopic Polytopic Proteolysis Protease Proteinase K Carboxypeptidase Y
Work in EC laboratory is supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02 and ANR-15-CE11-0019-01). MSV is a recipient of a doctoral fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche.
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