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Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Das, Mili and Kobayashi, Masanori and Yamada, Yusuke and Sreeramulu, Sridhar and Ramakrishnan, C and Wakatsuki, Soichi and Kato, Ryuichi and Varadarajan, Raghavan (2007) Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts. In: Journal of Molecular Biology, 372 (5). pp. 1278-1292.

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Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.

Item Type: Journal Article
Publication: Journal of Molecular Biology
Publisher: Elsevier
Additional Information: Copyright of this article belongs to Elsevier.
Keywords: protein polymers;intermolecular disulfides;protein crystallization;protein stability.
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 29 Jul 2008
Last Modified: 19 Sep 2010 04:48
URI: http://eprints.iisc.ac.in/id/eprint/15335

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