De novo design and characterization of copper centers in synthetic four-helix-bundle proteins

The design and chemical synthesis of de novo metalloproteins on cellulose m embranes with the structure of an antiparallel four-helix bundle is describ ed. All possible combinations of three different sets of amphiphilic helice s were assembled on cyclic peptide templates which were bound by a cleavabl e linker to the cellulose. In the hydrophobic interior, the four-helix bund le proteins carry a cysteine and several histidines at various positions fo r copper ligation. This approach was used successfully to synthesize, for t he first time, copper proteins based on a four-helix. bundle. UV-vis spectr a monitored on the solid support showed ligation of copper(II) by about one -third out of the 96 synthesized proteins and tetrahedral complexes of coba lt(II) by most of these proteins. Three of the most stable copper-binding p roteins were synthesized in solution and their structural properties analyz ed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and s ize-exclusion chromatography indicate a folding into a compact state contai ning a high degree of secondary structure with a reasonably ordered hydroph obic core. They displayed UV-vis absorption, resonance Raman, and EPR spect ra intermediate between those of type and type 2 copper centers. The presen t approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry gui ded by rational principles.