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  Thioredoxin as a fusion tag for carrier-driven crystallization

Corsini, L., Hothorn, M., Scheffzek, K., Sattler, M., & Stier, G. (2008). Thioredoxin as a fusion tag for carrier-driven crystallization. Protein Science, 17(12), 2070-2079. doi:10.1110/ps.037564.108.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002E-8C03-3 Version Permalink: http://hdl.handle.net/21.11116/0000-0000-E3A5-5
Genre: Journal Article

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ProtSci_17_2008_2070.pdf (Any fulltext), 727KB
 
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 Creators:
Corsini, Lorenzo, Author
Hothorn, Michael, Author
Scheffzek, Klaus1, Author              
Sattler, Michael, Author
Stier, Gunter2, Author              
Affiliations:
1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              
2Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: carrier-driven crystallization; fusion tag; Puf60; thioredoxin; linker sequence; NMR relaxation
 Abstract: Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.

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Language(s): eng - English
 Dates: 2008-07-162008-09-032009-01-022008-12
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
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Title: Protein Science
Source Genre: Journal
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Publ. Info: New York, N.Y. : Cambridge University Press
Pages: - Volume / Issue: 17 (12) Sequence Number: - Start / End Page: 2070 - 2079 Identifier: ISSN: 0961-8368
CoNE: /journals/resource/954925342760