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 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.
Department of Biochemistry & Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Diana M
DM
Mitrea
Lee S
LS
Parsons
Stewart N
SN
Loh
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D012260:Ribonucleases / Q000737:chemistry*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1