Molecular Biology Select

This issue's Molecular Biology Select highlights recent achievements in protein engineering. A new report describes deliberate modifications of a scaffold protein to control the output of the MAP kinase signaling pathway. Other recent studies illustrate several facets of rational protein design. These include the assembly of protein nanocapsules, an analysis of the determinants of virus particle stiffness, the creation of a new tag for protein visualization, and the use of genomic information to resurrect ancient proteins. The scaffold protein Ste5 assembles components of the MAP kinase (MAPK) pathway in yeast. New work by Bashor et al. (2008) shows how Ste5 can be deliberately engineered to control the output of the pathway. The authors created both positive and negative feedback loops by adding artificial binding sites (leucine zippers) to the existing Ste5 scaffold. The modified Ste5 proteins were then able to recruit regulators, which have been designed to include the complementary leucine zipper motif. To complete the feedback loops the expression of the engineered MAPK pathway regulators were themselves placed under the control of the MAPK pathway. For example, recruitment of the positive regulator of MAPK signaling, Ste50, enhanced transcriptional activity downstream of MAPK pathway activation and made the transcriptional response more switch-like in its behavior. In contrast, the recruitment to Ste5 of a negative regulator, the MAPK phosphatase Msg5, decreased transcriptional activation and flattened the dose-response curve. The magnitude of the effect can be further modulated by changing the strength of the interaction between the artificial binding site and the recruited protein. Using variations on this general scheme, the authors were able to engineer many different types of responses to MAPK signaling, including a ''pulse generator'' circuit, which peaks rapidly then tapers off, and an ultrasensitive switch that responds dramatically over a very narrow concentration of a-factor, an activator of the MAPK pathway. These findings not only demonstrate the critical and flexible roles of scaffolds in controlling signaling dynamics, but indicate strategies for the purposeful manipulation of cellular behavior for the emerging field of synthetic biology. Biocompatible nanocapsules might one day be used to selectively introduce drugs or other compounds into specific target cells. As a step in this direction, Ballister et al. (2008) now demonstrate the building of nanocapsules from a simple protein building block. In previous work, the authors determined the structure of Hcp1, a factor of unknown function secreted by the bacterium Pseudomonas aeruginosa. Hcp1 forms hexameric …