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.

Science 6 May 2005:
Vol. 308. no. 5723, pp. 857 - 860
DOI: 10.1126/science.1107387
Prev | Table of Contents | Next

Reports

Computational Thermostabilization of an Enzyme

Aaron Korkegian,1,2 Margaret E. Black,4 David Baker,3 Barry L. Stoddard1*

Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10°C increase in apparent melting temperature Tm and a 30-fold increase in half-life at 50°C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.

1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center (FHCRC), 1100 Fairview Avenue North, Seattle, WA 98109, USA.
2 Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, WA 98195, USA.
3 Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
4 Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164–6534, USA.

* To whom correspondence should be addressed. E-mail: bstoddar{at}fhcrc.org

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Protein design in biological networks: from manipulating the input to modifying the output.
A. M. Van der Sloot, C. Kiel, L. Serrano, and F. Stricher (2009)
Protein Eng. Des. Sel. 22, 537-542
   Abstract »    Full Text »    PDF »
Computational structure-based redesign of enzyme activity.
C.-Y. Chen, I. Georgiev, A. C. Anderson, and B. R. Donald (2009)
PNAS 106, 3764-3769
   Abstract »    Full Text »    PDF »
Rational stabilization of enzymes by computational redesign of surface charge-charge interactions.
A. V. Gribenko, M. M. Patel, J. Liu, S. A. McCallum, C. Wang, and G. I. Makhatadze (2009)
PNAS 106, 2601-2606
   Abstract »    Full Text »    PDF »
Rationally Designed Integrin {beta}3 Mutants Stabilized in the High Affinity Conformation.
B.-H. Luo, J. Karanicolas, L. D. Harmacek, D. Baker, and T. A. Springer (2009)
J. Biol. Chem. 284, 3917-3924
   Abstract »    Full Text »    PDF »
An antibody loop replacement design feasibility study and a loop-swapped dimer structure.
L. A. Clark, P. A. Boriack-Sjodin, E. Day, J. Eldredge, C. Fitch, M. Jarpe, S. Miller, Y. Li, K. Simon, and H. W.T. van Vlijmen (2009)
Protein Eng. Des. Sel. 22, 93-101
   Abstract »    Full Text »    PDF »
Thermostable Variants of Cocaine Esterase for Long-Time Protection against Cocaine Toxicity.
D. Gao, D. L. Narasimhan, J. Macdonald, R. Brim, M.-C. Ko, D. W. Landry, J. H. Woods, R. K. Sunahara, and C.-G. Zhan (2009)
Mol. Pharmacol. 75, 318-323
   Abstract »    Full Text »    PDF »
Thermostable variants constructed via the structure-guided consensus method also show increased stability in salts solutions and homogeneous aqueous-organic media.
E. Vazquez-Figueroa, V. Yeh, J.M. Broering, J.F. Chaparro-Riggers, and A.S. Bommarius (2008)
Protein Eng. Des. Sel. 21, 673-680
   Abstract »    Full Text »    PDF »
Optimal design of thermally stable proteins.
R. M. Bannen, V. Suresh, G. N. Phillips Jr, S. J. Wright, and J. C. Mitchell (2008)
Bioinformatics 24, 2339-2343
   Abstract »    Full Text »    PDF »
Bioinformatic method for protein thermal stabilization by structural entropy optimization.
E. Bae, R. M. Bannen, and G. N. Phillips Jr. (2008)
PNAS 105, 9594-9597
   Abstract »    Full Text »    PDF »
Algorithm for backrub motions in protein design.
I. Georgiev, D. Keedy, J. S. Richardson, D. C. Richardson, and B. R. Donald (2008)
Bioinformatics 24, i196-i204
   Abstract »    Full Text »    PDF »
Generation and Analysis of Mesophilic Variants of the Thermostable Archaeal I-DmoI Homing Endonuclease.
J. Prieto, J.-C. Epinat, P. Redondo, E. Ramos, D. Padro, F. Cedrone, G. Montoya, F. Paques, and F. J. Blanco (2008)
J. Biol. Chem. 283, 4364-4374
   Abstract »    Full Text »    PDF »
Four-Body Scoring Function for Mutagenesis.
C. Deutsch and B. Krishnamoorthy (2007)
Bioinformatics 23, 3009-3015
   Abstract »    Full Text »    PDF »
Biology by design: reduction and synthesis of cellular components and behaviour.
P. Marguet, F. Balagadde, C. Tan, and L. You (2007)
J R Soc Interface 4, 607-623
   Abstract »    Full Text »    PDF »
Optimal protein library design using recombination or point mutations based on sequence-based scoring functions.
R. J. Pantazes, M. C. Saraf, and C. D. Maranas (2007)
Protein Eng. Des. Sel.
   Abstract »    Full Text »    PDF »
Stabilization of Hyperactive Dihydrofolate Reductase by Cyanocysteine-mediated Backbone Cyclization.
H. Takahashi, M. Arai, T. Takenawa, H. Sota, Q. H. Xie, and M. Iwakura (2007)
J. Biol. Chem. 282, 9420-9429
   Abstract »    Full Text »    PDF »
Rapid detection of similarity in protein structure and function through contact metric distances.
A. M. Lisewski and O. Lichtarge (2006)
Nucleic Acids Res. 34, e152
   Abstract »    Full Text »    PDF »
Bioinformatics-driven, rational engineering of protein thermostability.
M. K. DiTursi, S.-J. Kwon, P. J. Reeder, and J. S. Dordick (2006)
Protein Eng. Des. Sel. 19, 517-524
   Abstract »    Full Text »    PDF »
RosettaDesign server for protein design..
Y. Liu and B. Kuhlman (2006)
Nucleic Acids Res. 34, W235-W238
   Abstract »    Full Text »    PDF »
Prediction and design of macromolecular structures and interactions.
D. Baker (2006)
Phil Trans R Soc B 361, 459-463
   Abstract »    Full Text »    PDF »
Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme.
F. GUO, A. R. GOODING, and T. R. CECH (2006)
RNA 12, 387-395
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)