“…drofolate reductase (26), aspartate transcarbamoylase (27,31), E. coli DsbA (28), glucose transporter (30), Bacillus -glucanase (49), and spectrin (50), has revealed that circularly permuted polypeptide chains can fold into stable and active proteins, albeit with, possibly, different topologies from those of the natural proteins. Most of the proteins so far circularly permuted (slightly over 20) have been small monomers, which have required linkers connecting the naturally close N and C termini in order to yield properly folded and active proteins (23,28,29,50,51). The characterization of active, circularly permuted ALAS variants, obtained through functional screening of a library of randomly circularized ALAS variants without linkers between their termini, has permitted us to conclude that circularly permuted ALAS variants are capable of folding into functional, native-like conformations, and thus that the natural location of the termini and sequential arrangement of the secondary structure elements are not critical determinants of the final protein fold, PLP cofactor attachment, or assembly of the two subunits into an active, dimeric enzyme.…”