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FOR THE RECORD

NMR spectroscopy as a tool for the rapid assessment of the conformation of GST-fusion proteins

School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia

(RECEIVED February 17, 2008; FINAL REVISION April 22, 2008; ACCEPTED April 23, 2008)

Glutathione-S-transferase (GST)-fusion proteins are used extensively for structural, biochemical, and functional analyses. Although the conformation of the target protein is of critical importance, confirmation of the folded state of the target is often not undertaken or is cumbersome because of the requirement to first remove the GST tag. Here, we demonstrate that it is possible to record conventional ¹⁵N-HSQC NMR spectra of small GST-fusion proteins and that the observed signals arise almost exclusively from the target protein. This approach constitutes a rapid and straightforward means of assessing the conformation of a GST-fusion protein without having to cleave the GST and should prove valuable, both to biochemists seeking to check the conformation of their proteins prior to functional studies and to structural biologists screening protein constructs for suitability as targets for structural studies.

Keywords: NMR spectroscopy; GST fusion; protein conformation; GST pulldown

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