Molten globules move into action.

O ne of the important goals of protein engineering and design is to create proteins with novel binding specificities. In addition to the intrinsic interest in understanding how the affinity and specificity of protein–ligand interactions is modulated, one can imagine a variety of practical applications for such a technology. The most relevant natural proteins are antibodies, in which an incredible range of binding specificities is displayed on the same basic scaffold. The specificity and affinity of antibodies for different target molecules is amazing, and yet to be matched by human design. In addition to the role for which antibodies naturally evolved, protection of the host organism from a wide range of invading pathogens, they are used in a variety of biotechnological applications such as affinity purification, in situ localization, immunoprecipitation, immunoblotting, and many others. Yet, despite the awesome properties of antibodies, they are not without problems. Monoclonal antibodies can be difficult and time-consuming to produce, animals must be killed, and the antibody protein is of high molecular weight and quite delicate in its storage and handling requirements. It would therefore be very useful if one could create a different protein scaffold, with none of the intrinsic problems of the antibody molecule, yet which could exhibit the positive binding properties of antibodies. How can this be done? One needs a means by which to select or screen for proteins that display the binding properties of interest. Although there are several emerging strategies for such in vitro selections, the most widely used to date has been ‘‘phage display.’’ The essential component of any selection strategy is that the genotype must be tied to phenotype. That is, when a protein, which displays a particular binding specificity, is selected for, there must be some way to know what changes in the protein have occurred and to obtain a ‘‘clone’’ of that protein. With monoclonal antibody production, the desired activity is screened for in monoclonal cell lines, which naturally contain the DNA encoding the antibody of interest, and the desired ‘‘clone’’ can be propagated. In phage display the protein of interest is ‘‘displayed’’ on the surface of the phage, as a fusion to one of the phage’s own coat proteins. The phage particle contains the DNA encoding the fusion protein and is thus tagged. Single-chain antibodies, Fv domains, and other engineered small fragments of antibodies have been displayed in this fashion on the surface of phage. The companion papers by Wahlberg et al. and Högbom et al. in a recent issue of PNAS (1, 2) take the strategy a step further. They chose a small, robust, well characterized protein and used phage display to evolve this molecule to have specific protein-binding activity. These papers describe the properties of a variant of the Z domain of staphylococcal protein A that was selected to bind to its parent, wild-type Z domain