Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N-and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k cat ∕K M by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k cat ∕K M by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.mutually exclusive folding | design | HIV protease | molecular switch | barnase Z ymogens are inactive enzyme precursors that are activated by proteolytic cleavage. Natural zymogens play essential roles in apoptosis, blood coagulation, digestion, viral maturation, and other cellular processes. Artificial zymogens are of broad interest, with potential applications that include diagnostic tools and toxins that attack pathogens or diseased cells. Here we introduce a unique technique (alternate frame protein folding, or AFF) to convert the cytotoxic ribonuclease barnase (Bn) into an artificial zymogen that is activated by HIV-1 protease (PR). The significance of AFF is that it is general and can thus be applied to many enzymes, including those whose activities are not regulated by any existing mechanism.Several groups have previously engineered artificial zymogens. Raines and colleagues circularly permuted ribonuclease A (RNase A) and bridged the original N-and C-termini with a peptide linker that contained cleavage sequences for PR, the NS3 protease from hepatitis C, and plasmepsin II from Plasmodium falciparium (1-3). The linkers inhibited enzymatic activity by partially occluding the active site. Cleavage with the respective protease increased k cat ∕K M by approximately 100-fold. In a recent study we took a related approach by permuting Bn with a short linker peptide intended to force the termini together, thereby distorting the active site or unfolding the enzyme (4). Cleaving the linker with a chemical reagent relieved conformational strain and increased RNase activity. Although these strategies successfully created artificial zymogens, they rely on specific properties of the target enzyme and are therefore not general. Cyclic permutation typic...