Engineering an artificial zymogen by alternate frame protein folding

Diana M. Mitrea, Lee S. Parsons, Stewart N. Loh

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


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 kcat/KM by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases kcat/KM 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.

Original languageEnglish (US)
Pages (from-to)2824-2829
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number7
StatePublished - Feb 16 2010


  • Design
  • HIV protease
  • Molecular switch
  • Mutually exclusive folding


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