Mapping functional sites on biological macromolecules

Electron microscopy of macromolecules dried from glycerol and rotary shadowed from a low angle can reveal the structure of individual molecules, or groups of molecules, with remarkable clarity. We used this technique to examine the interaction of the red blood cell cytoskeletal proteins spectrin, a 500,000 dalton protein which is long (750 A) and flexible;actin, a 43,000 dalton protein capable of polymerizing into double helical filaments; and band 4.1, an 82,000 dalton globular protein. By examining binary and ternary complexes of these molecules, the binding sites for actin, band 4.1 and a fourth protein ankyrin, which links the cytoskeleton to the membrane, have been mapped along the length of the spectrin molecule. These findings, which have enabled us to construct a model of the red cell cytoskeleton, show that low angle shadowing is a powerful but simple method for investigating associations among macromolecules.