A HYBRID SEQUENCE APPROACH TO THE PARACELSUS CHALLENGE

Inspired by the Paracelsus Challenge of nose and Creamer (Proteins 19: 1-3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold irate a largely bet a-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical ''parent'' prote in (434 Cro) and a ''target'' protein with the desired fold (the B1 do main of protein G), The hybrid sequence (Crotein-G) is 50% identical t o 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a poten tial His(3)Cys(1) zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circul ar dichroism spectra of the designed proteins are distinct from that o f 434 Cro and similar to that of the B1 domain of protein G. son ever, the proteins fail to denature in a cooperative manner. Furthermore, a ggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure for mation. In summary, 434 Cro has been altered to something that may res emble the Bi domain of protein G, but the protein does not adopt a nat ive structure. (C) 1998 Wiley-Liss, Inc.