PEDS Advance Access published online on October 14, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh079
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1 Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, the Netherlands
* To whom correspondence should be addressed. E-mail: w.j.quax{at}farm.rug.nl.
Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this study we show the redesign of a therapeutically interesting trimeric all beta-sheet protein, the cytokine TRAIL, yielding variants with improved thermal stability. A combination of TNF ligand family alignment information and the computational design algorithm, PERLA, were used to propose several mutants with improved thermal stability. The design was focused on non-conserved residues only, thus reducing use of computational resources. Several of the proposed mutants showed a significant increase in thermal stability as experimentally monitored by far-UV CD thermal denaturation. Stabilization of the biologically active trimer was achieved by monomer subunit or monomer-monomer interface modifications. A double mutant showed an increase in apparent Tm of 8 °C in comparison to wild-type TRAIL and remained biologically active after incubation at 73 °C for 1h. To our knowledge, this is the first study that improves the stability of a large multimeric
Revised September 24, 2004
Accepted October 5, 2004
Article
Stabilization of TRAIL, an all
-sheet multimeric protein, using computational redesign
2 Structural Biology and Biocomputing Program, EMBL, Meyerhofstrasse 1, D-69117, Heidelberg, Germany
Abstract 
-sheet protein structure by computational redesign. A similar approach can be used to alter the characteristics of other multimeric proteins, including other TNF ligand family members.
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