Transmembrane protein characterisation

Transmembrane proteins are key determinants of the pharmacokinetics of drugs and their characterisation is of increasing importance for the pharmaceutical industry.

Transmembrane proteins are a class of integral proteins that penetrate into or through the membrane bilayer from the outside to the inside of the cell. Transmembrane proteins account for more than 25% of proteins in complete genomes and are the targets for over 75% of pharmaceuticals.

The proteins have three regions or domains that can be defined: the domain in the bilayer, the domain outside the cell (called the extracellular domain), and the domain inside the cell (called the intercellular domain). Even though a cell membrane is somewhat fluid, the orientation of transmembrane proteins does not change. The proteins are so large that the rate for them to change orientation is extremely small. Thus, the extracellular part of the transmembrane protein is always outside the cell and the intercellular portion is always inside.

The lipid molecules of the membrane bilayer are predominantly hydrophobic and so the portion of the transmembrane protein that is embedded in the bilayer must have residues that are not polar. Commonly, these residues form a helix that is hydrophobic and therefore stable within the bilayer.

Transmembrane proteins play several roles in the functioning of cells. Communication is one of the most important roles: The proteins are useful for signalling to the cell what the external environment contains. Receptors are capable of interacting with specific substrate molecules on the extracellular domain. Once a protein binds to substrate, a change in the geometry near the binding site results in subsequent changes in the structure of the intercellular domain. These changes result in a cascade effect - another protein in the cell changes, affecting the next protein, and so on. Thus, transmembrane proteins are capable of initiating signals that are responsive to the external environment of the cell but ultimately lead to actions that take place in other structures of the cell.

In addition to serving as a way for the cell to gather information about the external environment, transmembrane proteins are associated with controlling the exchange of materials across the membrane. The proteins most involved in this process are called porins. These molecules appear in clusters that create channels within the membrane. In many cases the channels are regulated by other proteins so that they are open under some circumstances and closed under others.

However, the presence of hydrophobic transmembrane domain can result in:

• Low levels of expression
• Difficulties in solubilisation
• Difficulties in crystallisation

Attempting crystallisation and structure solution of transmembrane proteins is considered difficult and risky.

The Université de Provence and Université de la Méditerranée in Marseille, France, has implemented the Wyatt multi-angle light scattering (MALS) detectors for use in their Biological Macromolecules Joint Research Unit. The instruments are used for the characterisation of transmembrane proteins, determining their quaternary structure and following their retention during protein handling.

Wyatt’s MALS detectors offer an easy-to-use, efficient alternative to traditional laborious in vitro characterisation techniques.

Conventional in vitro characterisation of transmembrane proteins requires the extraction of the proteins from the membrane and subsequent maintenance in a soluble and native state. This is generally achieved using amphiphilic compounds, termed detergents, yielding protein-detergent complexes (PDC). However, several transmembrane proteins are naturally found as oligomers and maintaining this precise macromolecular assembly is a crucial issue for further studies. In addition, classical SEC column calibration does not apply in the case of PDC, whose volume and shape depends on the detergent fraction.

For this particular application, Wyatt’s MALS instruments were used to perform a quaternary structure study of the Methanosarcina mazei CorA transporter in two detergents. MALS technology was used in conjunction with refractometry and UV280 nm absorbance. This combined solution was able to solve a ‘two equations with two unknown parameters’ system, providing masses of protein and detergent in each PDC. This approach provided hints about the retention of the native, and thus active, membrane protein quaternary structure, which is a crucial issue for crystallisation or other biochemical studies, without performing laborious activity tests.

Experimental results prove that Wyatt’s MALS instrumentation surpasses traditional in vitro characterisation techniques, providing a straightforward method capable of accurate, reliable characterisation of transmembrane proteins. The capabilities of Wyatt’s MALS detectors for characterising transmembrane proteins are illustrated in an application note, entitled Membrane Protein Quaternary Structure Analysis and Detergent Selection, which is available to download from www.wyatt.com.