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January 15, 2009 (Vol. 29, No. 2)

Tools for Studying Membrane Protein Targets

Technology Useful for High-Throughput Screening and Lead-Optimization Projects

  • Approximately 40% of today’s pharmaceuticals act on proteins such as GPCRs  and ion channels that are embedded within cell membranes. These transmembrane proteins form the functional environmental interface of the cell, accounting for their value as therapeutic targets. They have been exploited to treat a wide range of diseases, including cancer, heart disease, diabetes, nervous system disorders, and infectious diseases.

    Studying membrane protein structure and interactions during drug characterization and optimization, however, presents a unique set of challenges. Typically, membrane protein structural conformation is dependent on the presence of the cellular lipid membrane. Conventional binding assays for membrane proteins employ live cells or membrane preparations derived from cells to ensure structural stability of the embedded target proteins.

    These formats, however, exhibit low target protein concentration, receptor heterogeneity, lipid contaminants, and, in the case of membrane preparations, receptor inversion, leading to poor sensitivity and high experimental variability. Furthermore, because of their large size and heterogeneity, cells and membrane vesicles are not amenable to advanced detection devices that employ microfluidic systems such as optical biosensors.

    A number of novel strategies have attempted to isolate membrane proteins from the lipid bilayer of the cell and present them in a high concentration, homogeneous format. Most techniques involve the reconstitution of receptors onto artificial structures, such as beads or micelles. These methods use detergents to control protein unfolding and micelle incorporation. Since detergents and artificial lipids can alter membrane protein structure, conditions must be determined empirically and validated for each membrane protein studied.

    An alternative to detergent-based manipulation is to express membrane proteins in biological particles, such as intracellular compartments of specialized bacteria. While bacterial systems offer the benefits of high concentration protein expression, target proteins must typically be fused with native anchor proteins to ensure trafficking to membrane surfaces. The lack of mammalian post-translational modifications and the differences in cell membrane composition can also influence protein structure and function.

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