Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis (protein fodig/protein secretion/x-ray crystaflography/circular permutation)

A jellyroll a-sandwich protein, the Bacilus Pgluanase H(A16-M), is used to probe the role ofN-terminal peptide regions in protein folding in vivo. A gene e in H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214. The rearranged gene is expressed in Escherichia coli. The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stableand activeenzyme. Crystal s e analysis at 2.0- resolution, R = 15.3%, showscpAl6M-59 tohave a three-dimensional structure nearly identical with that of the parent figlucanase. An anogous experiment based on the wild-type Bacillus nacerans 3-fluca- nase, giving rise to the circularly permuted variantcpMAC-57, yields the ame results. Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted