Functional proteins from designed combinatorial libraries

The ability to harness the enzymatic power of proteins through the de novo design of functional sequences is a long-standing goal of protein design. A combinatorial library of sequences targeted to fold into four-helix bundles was previously constructed using a “binary code” strategy, in which the patterning of polar and nonpolar amino acids was specified explicitly, but the exact identities of the side chains were varied extensively. The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. This collection of novel heme proteins provided a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection.; To compare this unbiased collection of heme proteins to natural heme proteins, a series of experiments were performed. (1) The catalytic potential was sampled by screening roughly 30 of the heme proteins for peroxidase activity. Several candidates showed significant levels of peroxidase activity, with the best candidate having turnover numbers roughly one-third that of Horseradish Peroxidase. (2) A subset of the heme-protein collection was analyzed for carbon monoxide binding. The affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of eight of the designed heme proteins were measured. The designed heme proteins had CO binding and release rates and Raman spectra similar to natural heme proteins such as myoglobin and hemoglobin. (3) A subset of the heme proteins was analyzed to determine both the heme to protein binding stoichiometry and the midpoint redox potential.; In an attempt to actively select for ligand binding functionality, a second library of protein sequences was constructed and screened. This library was previously designed by Dan Rosenbaum and Michael Hecht to fold into a-helical coiled coil proteins. The sequences were prepared as fusion proteins to the major coat protein of M13 bacteriophage for use in screening as a phage-displayed peptide library. This library contains approximately six million different protein sequences and has been used to screen for heme binding interactions.