Document Detail


Engineering an artificial zymogen by alternate frame protein folding.
MedLine Citation:
PMID:  20133757     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Diana M Mitrea; Lee S Parsons; Stewart N Loh
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-01-26
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  107     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2010 Feb 
Date Detail:
Created Date:  2010-02-17     Completed Date:  2010-05-10     Revised Date:  2014-09-21    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2824-9     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Enzyme Precursors / biosynthesis*,  genetics,  metabolism
HIV Protease / metabolism*
Models, Molecular*
Molecular Sequence Data
Protein Conformation*
Protein Engineering / methods*
Protein Folding*
Ribonucleases / chemistry*
Grant Support
ID/Acronym/Agency:
GM069755/GM/NIGMS NIH HHS; R01 GM069755/GM/NIGMS NIH HHS; R01 GM069755-05A1/GM/NIGMS NIH HHS; R21 AI068440/AI/NIAID NIH HHS; R21 AI068440-01A2/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Enzyme Precursors; EC 3.1.-/Ribonucleases; EC 3.1.4.-/Bacillus amyloliquefaciens ribonuclease; EC 3.4.23.-/HIV Protease; EC 3.4.23.-/p16 protease, Human immunodeficiency virus 1
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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