Stabilizing membrane proteins through protein engineering

• Integral membrane proteins (IMPs) are generally unstable when removed from membranes.
• This instability impedes structural and biochemical characterization of most IMPs.
• The detergent stability of IMPs can be improved by the addition of stabilizing mutations.
• Stabilized IMPs can be used for structural, biophysical and biochemical investigations.
• Identifying stabilizing mutations is difficult and current methods to find them are discussed.

Integral membrane proteins (IMPs) are crucial components of all cells but are difficult to study in vitro because they are generally unstable when removed from their native membranes using detergents. Despite the major biomedical relevance of IMPs, less than 1% of Protein Data Bank (PDB) entries are IMP structures, reflecting the technical gap between studies of soluble proteins compared to IMPs. Stability can be engineered into IMPs by inserting stabilizing mutations, thereby generating proteins that can be successfully applied to biochemical and structural studies when solubilized in detergent micelles. The identification of stabilizing mutations is not trivial, and this review will focus on the methods that have been used to identify stabilized membrane proteins, including alanine scanning and screening, directed evolution and computational design.