Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Feb 16;107(7):2824-9.
doi: 10.1073/pnas.0907668107. Epub 2010 Jan 26.

Engineering an artificial zymogen by alternate frame protein folding

Diana M Mitrea  1 , Lee S Parsons, Stewart N Loh
Affiliations

Engineering an artificial zymogen by alternate frame protein folding

Diana M Mitrea et al. Proc Natl Acad Sci U S A. .

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
X-ray crystal structure of WT Bn. The dark gray region corresponds to the C-terminal segment of Bn-AFF that is duplicated and fused to the N-terminus. Amino acid positions 1 (N-terminus), 93 (site of circular permutation), 102 (catalytic acid; alpha carbon shown), and 110 (C-terminus) are indicated.
Fig. 2.
Fig. 2.
Amino acid sequences of Bn variants and mechanism of AFF switching. (A) Amino acid sequences of enzymes created in this study. Numbering is according to the WT Bn sequence; prime superscripts indicate amino acids in the F2 segment of Bn-AFF. Duplicate segments are shown in red and blue. Open and closed circles denote the presence of His102 or Ala102, respectively. Dashed lines represent the PR cleavage site (IFLETS). Solid lines symbolize the flexible linker used to bridge the original N- and C-termini in Bn-F2 and Bn-AFF. (B) Schematic illustrating the topological change that underlies the F1⇆F2 fold shift. F1 and F2 analogs are shown. Dashed line and open/closed circles are the same as in panel A. (C) Schematic of AFF switching mechanism. Wavy lines indicate unfolded regions; colors, dashed line, and open/closed circles are the same as in panel A.
Fig. 3.
Fig. 3.
CD spectra of Bn-AFF variants and their F1/F2 analogs. Lines are as follows: Bn-F1 (Solid Gray), Bn-F2 (Dashed Gray), Bn-F2(P94) (Dotted Black), Bn-AFF(P94 + A96) (Dashed Black), Bn-AFF(P94′) (Solid Black).
Fig. 4.
Fig. 4.
Urea-induced denaturation of Bn-AFF before stability optimization (A), and after stability optimization (B). Symbols in panel A are: Bn-F1 (Open Circles), Bn-AFF (Closed Circles), Bn-F2 (Triangles). Symbols in panel B are: Bn-F1 (Open Circles), Bn-AFF(P94′) (Closed Squares), Bn-F2(P94) (Inverted Triangles). Lines are best fits of the data to the linear extrapolation equation.
Fig. 5.
Fig. 5.
Limited trypsin digestion of Bn-AFF(P94′) characterized by mass spectrometry. Peaks marked with an asterisk or +2 indicate an impurity or the +2 charge species of a parent peak, respectively. Samples are as follows: Bn-AFF(P94′) (A), Bn-AFF(P94′) + trypsin (B), PR-digested Bn-AFF(P94′) (C), PR-digested Bn-AFF(P94′) + trypsin (D).
Fig. 6.
Fig. 6.
Hydrolysis of the fluorogenic RNA substrate 5′ 6-FAM-ArGAA-3′TAMRA by Bn-AFF(P94′). Lines are best fits of the data to equations in ref. .
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Plainkum P, Fuchs SM, Wiyakrutta S, Raines RT. Creation of a zymogen. Nat Struct Biol. 2003;10:115–119. - PMC - PubMed
    1. Turcotte RF, Raines RT. Design and characterization of an HIV-specific ribonuclease zymogen. AIDS Res Hum Retro. 2008;24:1357–1363. - PMC - PubMed
    1. Johnson RJ, Lin SR, Raines RT. A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus. FEBS J. 2006;273:5457–5465. - PubMed
    1. Butler JS, Mitrea DM, Mitrousis G, Cingolani G, Loh SN. Structural and thermodynamic analysis of a conformationally-strained circular permutant of barnase. Biochemistry. 2009;48:3497–3507. - PMC - PubMed
    1. Mossakowska DE, Nyberg K, Fersht AR. Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquifaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry. 1989;28:3843–3850. - PubMed

Publication types

LinkOut - more resources