Probing Protein Topology and Conformation by Limited Proteolysis

Proteases are enzymes that catalyze the hydrolytic degradation of other proteins into peptides or amino acids through the digestion of the peptide bond. Promiscuous proteases that target a wide range of proteins are distinguished from specific proteases that have a narrow range of substrates. In terms of activity, endoproteases cleave their substrates at specific residues within the target proteins, whereas exoproteases cleave from one extremity and may have processive activities. Proteases are therefore very useful tools to study proteins, notably their structure or conformation. In addition, proteases can be used to probe the topology of bacterial membrane proteins. Here, we describe limited protease accessibility assays to define inner membrane protein topology and conformational changes based on digestion profiles.