Many membrane proteins function as part of multicomponent complexes, several of which have been studied by x-ray crystallography. One is a bacterial photosynthetic reaction center, which was the first membrane protein complex to be crystallized and analyzed by x-ray diffraction. In Chapter 14, we discuss how such photosynthetic complexes function to capture light energy and use it to pump H+ across the membrane. Many of the membrane protein complexes involved in photosynthesis, proton pumping, and electron transport are even larger than the photosynthetic reaction center. The enormous photosystem II complex from cyanobacteria, for example, contains 19 protein subunits and well over 60 transmembrane helices (see Figure 14–49). Membrane proteins are often arranged in large complexes, not only for harvesting various forms of energy, but also for transducing extracellular signals into intracellular ones (discussed in Chapter 15).
Many Membrane proteins Diffuse in the plane of the Membrane
Like most membrane lipids, membrane proteins do not tumble (flip-flop) across the lipid bilayer, but they do rotate about an axis perpendicular to the plane of the bilayer (rotational diffusion). In addition, many membrane proteins are able to move laterally within the membrane (lateral diffusion). An experiment in which mouse cells were artificially fused with human cells to produce hybrid cells (heterokaryons) provided the first direct evidence that some plasma membrane proteins are mobile in the plane of the membrane. Two differently labeled antibodies were used to distinguish selected mouse and human plasma membrane proteins. Although at first the mouse and human proteins were confined to their own halves of the newly formed heterokaryon, the two sets of proteins diffused and mixed over the entire cell surface in about half an hour (Figure 10–32).
The lateral diffusion rates of membrane proteins can be measured by using the technique of fluorescence recovery after photobleaching (FRAP). The method usually involves marking the membrane protein of interest with a specific fluorescent group. This can be done either with a fluorescent ligand such as a fluorophore-labeled antibody that binds to the protein or with recombinant DNA technology to express the protein fused to a fluorescent protein such as green fluorescent protein (GFP) (discussed in Chapter 9). The fluorescent group is then bleached in a small area of membrane by a laser beam, and the time taken for adjacent membrane proteins carrying unbleached ligand or GFP to diffuse into the bleached area is measured (Figure 10–33). From FRAP measurements, we can estimate the diffusion coefficient for the marked cell-surface protein. The values of the diffusion coefficients for different membrane proteins in different cells are highly variable, because interactions with other proteins impede the diffusion of the proteins to varying degrees. Measurements of proteins that are minimally impeded in this way indicate that cell membranes have a viscosity comparable to that of olive oil.
One drawback to the FRAP technique is that it monitors the movement of large populations of molecules in a relatively large area of membrane; one cannot follow individual protein molecules. If a protein fails to migrate into a bleached area, for example, one cannot tell whether the molecule is truly immobile or just restricted in its movement to a very small region of membrane—perhaps by cytoskeletal proteins. Single-particle tracking techniques overcome this problem by labeling individual membrane molecules with antibodies coupled to fluorescent dyes or tiny gold particles and tracking their movement by video microscopy. Using single-particle tracking, one can record the diffusion path of a single membrane protein molecule over time. Results from all of these techniques indicate that plasma membrane proteins differ widely in their diffusion characteristics, as we now discuss.