NMR spectroscopy as a tool for the rapid assessment of the conformation of GST-fusion proteins.
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 (15)N-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.
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia. ckliew@mmb.usyd.edu.au
Chu Kong
CK
Liew
Roland
R
Gamsjaeger
Robyn E
RE
Mansfield
Joel P
JP
Mackay
D000818:Animals; D002021:Buffers; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D005069:Evaluation Studies as Topic; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors
D002021:Buffers; D029742:Caenorhabditis elegans Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase