DE-NOVO DESIGN OF THE HYDROPHOBIC CORES OF PROTEINS

We have developed and experimentally tested a novel computational appr oach for the de novo design of hydrophobic cores. A pair of computer p rograms has been written, the first of which creates a ''custom'' rota mer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program uses a geneti c algorithm to globally optimize for a low energy core sequence and st ructure, using the custom rotamer library as input. Success of the pro grams in predicting the sequences of native proteins indicates that th ey should be effective tools for protein design. Using these programs, we have designed and engineered several variants of the phage 434 cro protein, containing five, seven, or eight sequence changes in the hyd rophobic core. As controls, we have produced a variant consisting of a randomly generated core with six sequence changes but equal volume re lative to the native core and a variant with a ''minimalist'' core con taining predominantly leucine residues. Two of the designs, including one with eight core sequence changes, have thermal stabilities compara ble to the native protein, whereas the third design and the minimalist protein are significantly destabilized. The randomly designed control is completely unfolded under equivalent conditions. These results sug gest that rational de novo design of hydrophobic cores is feasible, an d stress the importance of specific packing interactions for the stabi lity of proteins. A surprising aspect of the results is that all of th e variants display highly cooperative thermal denaturation curves and reasonably dispersed NMR spectra. This suggests that the non-core resi dues of a protein play a significant role in determining the uniquenes s of the folded structure.