Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization.

Selection and characterization of DARPins specific for the neurotensin receptor 1.

We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat neurotensin receptor 1 (NTR1), a G-protein coupled receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 microg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy.