DESIGN, EXPRESSION, AND INITIAL CHARACTERIZATION OF M1, A DE-NOVO PROTEIN ENRICHED IN ESSENTIAL AMINO-ACIDS

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, l ysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino ac ids, that would adopt the alpha-helical bundle fold. The design proces s was an iterative one. The consensus residues (based on the propertie s profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-t ermini. Using these data, the helices in our de novo protein were desi gned by inserting tbe residues M, T, K and L as often as possible at p ositions where their volume, hydrophobicity and charge match the conse nsus found in natural bundle helices. Short sequences of strong turn f ormers were used to join the helices and adjust the predicted pI to 7. 7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known p rotease targets in E. coli. The sequence of our de novo protein, MB1, is: MTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA, where dashes se parate long helices from short, turn forming linkers. A gene coding fo r this protein was assembled from synthetic oligonucleotides, then fus ed to the maltose binding protein gene under the control of a tac prom oter. The fusion protein was expressed in E. coli. purified and cleave d to yield maltose binding protein and our de novo protein, MB1. MB1 w as found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K a nd L.