DE-NOVO DESIGN OF REDOX PROTEINS

A modular strategy for the de-novo synthesis of four-helix bundle prot eins has been established. The individual helices and a cyclic decapep tide are purified to homogeneity after deprotection before they are co upled chemo- and regioselectively in predetermined orientations. The o rientation of the helices relative to each other as well as of the N-t erminus is controlled by different side chain protecting groups. The n ew protein adapts the predicted structure of the heme binding core of the transmembrane cytochrome b to a soluble form. This protein with a molecular mass of 14,293 with antiparallel orientation of the four hel ices binds two heme groups in its hydrophobic interior via four histid ines. Circular dichroism studies indicate a high helical content of th e complex and a free energy of folding of -30.4 kJ/mol. The two heme g roups have midpoint potentials of -106 and -170 mV, respectively. The modular design of the de-novo protein can easily be modified and provi des the basis for detailed physical studies of molecular parameters.