Getting membrane proteins into the fold

Membrane-embedded proteins execute many of the most important cellular functions, detecting external environmental cues, mediating communication and signaling, and facilitating molecular import and export. Accordingly, they also represent important targets for drug development, and an estimated 60% of currently available drugs are believed to target membrane proteins.

On the other hand, experimental investigation of the structure and function of individual membrane proteins is routinely thwarted by the general difficulty of preparing large quantities of properly folded protein. This is largely due to the chemical composition of these proteins, which have large hydrophobic surfaces that are chemically averse to being exposed to water; these protein segments are stable when embedded within the equally hydrophobic lipid-based cell membrane (Fig. 1), but tend to assemble into irregular, improperly folded clumps when prepared in solution.

Most water-soluble proteins can readily be produced either within modified cell lines or through the use of cellular extracts that contain the full complement of machinery required for protein synthesis. However, the yield of membrane proteins obtained from cultured cells is generally inadequate, and scientists have had to tinker extensively with extract-based production methods to obtain usable quantities of functional protein.

Now, a research team led by Shigeyuki Yokoyama at the RIKEN Systems and Structural Biology Center in Yokoyama has developed an efficient preparation method that produces high yields of functional membrane proteins¹. “Up until now, aggregation and misfolding of membrane proteins during cell-free protein synthesis have been avoided by adding either lipid-based liposomes or detergents,” explains Yokoyama. However, he adds that such workarounds were not a complete solution, and challenges remained in selecting the appropriate detergent for a given protein, or ensuring that newly synthesized proteins were properly folded and inserted into the membranes of liposomes.

Yokoyama and colleagues solved this problem by developing a hybrid technique that incorporates elements of both preparation methods, synthesizing their proteins in a bacteria-derived extract containing both detergent and lipid molecules. During production, hydrophobic stretches of the newly formed protein are protected by this lipid-detergent mixture; once synthesis is complete, the detergent is removed from the sample via dialysis and the remaining lipids subsequently assemble into bubble-shaped liposomes with the mature proteins securely embedded within these artificial membranes (Fig. 2).

The researchers then demonstrated the efficacy of their technique with bacteriorhodopsin (BR), an archaea-derived photosynthetic pigment protein. The light-responsive properties of BR made it a particularly useful test subject for this method—improperly folded or aggregated BR appears yellow, while preparations of mature BR appear purple. In initial experiments, they were able to demonstrate successful production of properly folded BR with a variety of detergents, and although the overall folding efficiency was somewhat lower than previously described preparations, the overall quantity of protein produced was significantly greater—as much as 80-fold greater, depending on the detergent. Subsequent analysis confirmed that much of this properly folded BR was successfully integrated into the membranes of liposomes following detergent removal.

Yokoyama and his colleagues' protein preparations also retained proper functional characteristics. Naturally occurring BR undergoes a series of chemical transitions known as a photocycle, in which excitation by light at particular wavelengths induces a series of subtle structural changes; these shifts can in turn be quantified by changes in the light-absorption properties of the protein. They found that their synthetic BR mirrored the photocyclic characteristics of native protein, and even displayed the capacity to act as a light-activated proton pump—a key component of its photosynthetic activity.