Many membrane proteins can be solubilized and then purified in an active form by the use of mild detergents. These detergents cover the hydrophobic regions on membrane-spanning segments that become exposed after lipid removal but do not unfold the protein. If the detergent concentration of a solution of solubilized membrane proteins is reduced (by dilution, for example), membrane proteins do not remain soluble. In the presence of an excess of phospholipid molecules in such a solution, however, membrane proteins incorporate into small liposomes that form spontaneously. In this way, functionally active membrane protein systems can be reconstituted from purified components, providing a powerful means of analyzing the activities of membrane transporters, ion channels, signaling receptors, and so on (Figure 10–28). Such functional reconstitution, for example, provided proof for the hypothesis that the enzymes that make
ADDITION OF PHOSPHOLIPIDS (mixed with
Figure 10–27 Solubilizing a membrane protein with a mild nonionic detergent.
the detergent disrupts the lipid bilayer and brings the protein into solution as protein–lipid–detergent complexes. the phospholipids in the membrane are also solubilized by the detergent, as lipid-detergent micelles.
Figure 10–28 The use of mild nonionic detergents for solubilizing, purifying, and reconstituting functional membrane protein systems. In this example, functional na+-K+ pump molecules are purified and incorporated into phospholipid vesicles. this pump is present in the plasma membrane of most animal cells, where it uses the energy of Atp hydrolysis to pump na+ out of the cell and K+ in, as discussed in chapter 11.
ATP (ATP synthases) use H+ gradients in mitochondrial, chloroplast, and bacterial membranes to produce ATP.
Membrane proteins can also be reconstituted from detergent solution into nanodiscs, which are small, uniformly sized patches of membrane that are surrounded by a belt of protein, which covers the exposed edge of the bilayer to keep the patch in solution (Figure 10–29). The belt is derived from high-density lipoproteins (HDL), which keep lipids soluble for transport in the blood. In nanodiscs the membrane protein of interest can be studied in its native lipid environment and is experimentally accessible from both sides of the bilayer, which is useful, for example, for ligand-binding experiments. Proteins contained in nanodiscs can also be analyzed by single particle electron microscopy techniques to determine their structure. By this rapidly improving technique (discussed in Chapter 9), the structure of a membrane protein can be determined to high resolution without a requirement of the protein of interest to crystallize into a regular lattice, which is often hard to achieve for membrane proteins.
Detergents have also played a crucial part in the purification and crystallization of membrane proteins. The development of new detergents and new expression systems that produce large quantities of membrane proteins from cDNA clones has led to a rapid increase in the number of three-dimensional structures of membrane proteins and protein complexes that are known, although they are still few compared to the known structures of water-soluble proteins and protein complexes.
Bacteriorhodopsin Is a light-driven proton (h+) pump that traverses the lipid Bilayer as Seven α helices
In Chapter 11, we consider how multipass transmembrane proteins mediate the selective transport of small hydrophilic molecules across cell membranes. But a detailed understanding of how such a membrane transport protein works requires precise information about its three-dimensional structure in the bilayer. Bacteriorhodopsin was the first membrane transport protein whose structure was determined, and it has remained the prototype of many multipass membrane proteins with a similar structure.