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Engineering an artificial zymogen by alternate frame protein folding
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...
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Published in: | Proceedings of the National Academy of Sciences - PNAS 2010-02, Vol.107 (7), p.2824-2829 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.0907668107 |