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Corsini, L.* ; Hothorn, M.* ; Scheffzek, K.* ; Sattler, M. ; Stier, G.*

Thioredoxin as a fusion tag for carrier-driven crystallization.

Protein Sci. 17, 2070-2079 (2008)
DOI
Open Access Green as soon as Postprint is submitted to ZB.
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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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords carrier-driven crystallization; fusion tag; Puf60; thioredoxin; linker sequence; NMR relaxation
ISSN (print) / ISBN 0961-8368
e-ISSN 1469-896X
Journal Protein Science
Quellenangaben Volume: 17, Issue: 12, Pages: 2070-2079 Article Number: , Supplement: ,
Publisher Wiley
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Structural Biology (STB)