Membrane Protein Stabilization Strategies for Structural and Functional Studies

Abstract

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

Keywords: antibody; detergent; ligand; lipid; membrane proteins; mutagenesis; nanobody; stability.

Figure 1

Schematic representation of the split GFP assay as reporter of membrane protein expression and stability in the membrane. The split GFP assay consists of two plasmids: one containing the target membrane protein fused to GFP strand 11 (left) and another plasmid expressing GFP strands 1 to 10 (right). Protein expression is controlled by two promoters activated by different inducers (I1 and I2). Inducing these two genes sequentially allows the identification of target membrane protein variants that are expressed and inserted into the plasma membrane of the expression system, since the two fragments of the GFP complements resulting in fluorescence emission. Variants confined into inclusion bodies show no fluorescence as no complementation occurs.

Figure 2

Membrane protein incorporation into a nanodisc. Detergent (black micelle)-solubilized membrane protein (blue) is incubated with a mixture of membrane scaffold protein (MSP; green) and detergent-solubilized lipids (tan). Nanodisc assembly is initiated after detergent removal.

Figure 3

Nanobodies in membrane protein structural biology. (a) Traditional monoclonal antibodies (mAb) and their fragments (Fab) vs. heavy chain antibodies (HcAb) and nanobodies (Nb). (b) Nanobody structure (PDB ID: 6f2g) [101], showing complementary determining regions (CDR) 1 (magenta), 2 (blue), and 3 (orange) and the disulfide bridge established between Cys 22 and Cys 96. (c) Extensive interactions were found between the side chains and backbones of the nanobody CDR3 (orange) and the bacterial amino acid transporter BasC (PDB ID: 6f2g) [101]. CDR3 interacts with residues from BasC transmembrane domains (TMs) 1, 6, 8 and 9.