Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
INT'L BIO FORUM & BIO EXPO JAPAN

Site Tools

  • AAAS
  • Subscribe
  • Feedback

Site Search

Search Advanced

Science 20 February 2004:
Vol. 303. no. 5661, pp. 1185 - 1189
DOI: 10.1126/science.1092612
Prev | Table of Contents | Next

Reports

An Engineered Pathway for the Formation of Protein Disulfide Bonds

Lluis Masip,1 Jonathan L. Pan,3,4 Suranjana Haldar,5 James E. Penner-Hahn,5 Matthew P. DeLisa,1 George Georgiou,1,2* James C. A. Bardwell,3,4* Jean-François Collet3

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

1 Department of Chemical Engineering and Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
2 Department of Biomedical Engineering, University of Texas, Austin, TX 78712, USA.
3 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
4 Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.
5 Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

* To whom correspondence should be addressed. E-mail: gg{at}che.utexas.edu (G.G.); jbardwel{at}umich.edu (J.C.A.B.)

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide: A NEW PARADIGM FOR BACTERIAL OXIDATIVE FOLDING.
B. Heras, M. Kurz, R. Jarrott, S. R. Shouldice, P. Frei, G. Robin, M. Cemazar, L. Thony-Meyer, R. Glockshuber, and J. L. Martin (2008)
J. Biol. Chem. 283, 4261-4271
   Abstract »    Full Text »    PDF »
Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant.
L. Masip, D. Klein-Marcuschamer, S. Quan, J. C. A. Bardwell, and G. Georgiou (2008)
J. Biol. Chem. 283, 840-848
   Abstract »    Full Text »    PDF »
Cysteine Scanning Mutagenesis and Topological Mapping of the Escherichia coli Twin-Arginine Translocase TatC Component.
C. Punginelli, B. Maldonado, S. Grahl, R. Jack, M. Alami, J. Schroder, B. C. Berks, and T. Palmer (2007)
J. Bacteriol. 189, 5482-5494
   Abstract »    Full Text »    PDF »
Laboratory evolution of one disulfide isomerase to resemble another.
A. Hiniker, G. Ren, B. Heras, Y. Zheng, S. Laurinec, R. W. Jobson, J. A. Stuckey, J. L. Martin, and J. C. A. Bardwell (2007)
PNAS 104, 11670-11675
   Abstract »    Full Text »    PDF »
Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1.
N. Rouhier, H. Unno, S. Bandyopadhyay, L. Masip, S.-K. Kim, M. Hirasawa, J. M. Gualberto, V. Lattard, M. Kusunoki, D. B. Knaff, et al. (2007)
PNAS 104, 7379-7384
   Abstract »    Full Text »    PDF »
Conservation and Variation between Rhodobacter capsulatus and Escherichia coli Tat Systems.
U. Lindenstrauss and T. Bruser (2006)
J. Bacteriol. 188, 7807-7814
   Abstract »    Full Text »    PDF »
Cysteine-scanning Mutagenesis and Disulfide Mapping Studies of the Conserved Domain of the Twin-arginine Translocase TatB Component.
P. A. Lee, G. L. Orriss, G. Buchanan, N. P. Greene, P. J. Bond, C. Punginelli, R. L. Jack, M. S. P. Sansom, B. C. Berks, and T. Palmer (2006)
J. Biol. Chem. 281, 34072-34085
   Abstract »    Full Text »    PDF »
The origami of thioredoxin-like folds..
J. L. Pan and J. C.A. Bardwell (2006)
Protein Sci. 15, 2217-2227
   Abstract »    Full Text »    PDF »
Conserved Role of the Linker {alpha}-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB.
L. Segatori, L. Murphy, S. Arredondo, H. Kadokura, H. Gilbert, J. Beckwith, and G. Georgiou (2006)
J. Biol. Chem. 281, 4911-4919
   Abstract »    Full Text »    PDF »
Effects of Disulfide Bonds on Folding Behavior and Mechanism of the {beta}-Sheet Protein Tendamistat.
M. Qin, J. Zhang, and W. Wang (2006)
Biophys. J. 90, 272-286
   Abstract »    Full Text »    PDF »
Identification of a Twin-Arginine Translocation System in Pseudomonas syringae pv. tomato DC3000 and Its Contribution to Pathogenicity and Fitness.
P. A. Bronstein, M. Marrichi, S. Cartinhour, D. J. Schneider, and M. P. DeLisa (2005)
J. Bacteriol. 187, 8450-8461
   Abstract »    Full Text »    PDF »
The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.
J.-F. Collet, D. Peisach, J. C.A. Bardwell, and Z. Xu (2005)
Protein Sci. 14, 1863-1869
   Abstract »    Full Text »    PDF »
Characterization of human glutaredoxin 2 as iron-sulfur protein: A possible role as redox sensor.
C. H. Lillig, C. Berndt, O. Vergnolle, M. E. Lonn, C. Hudemann, E. Bill, and A. Holmgren (2005)
PNAS 102, 8168-8173
   Abstract »    Full Text »    PDF »
Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways.
L. Segatori, P. J. Paukstelis, H. F. Gilbert, and G. Georgiou (2004)
PNAS 101, 10018-10023
   Abstract »    Full Text »    PDF »


ADVERTISEMENT
Click Me!

ADVERTISEMENT

To Advertise     Find Products