Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site.
Histidine (His) tags are one of the most popular fusion tags for the isolation of proteins via metal affinity chromatography. The fusion tag is routinely left attached to the protein when carrying out experiments, with the assumption that the addition has no effect on structure or function. In the present study, we have prepared four proteins of the gene regulatory protein AreA from Aspergillus nidulans for crystallization experiments: a 91-amino acid peptide encompassing the minimal DNA-binding region, both with and without the His-tag (HZFB and ZFB, respectively), and a 155-amino acid protein previously proposed to be the entire DNA-binding domain for AreA, both with and without the His-tag (HG1b and G1b, respectively). To test the integrity of the four AreA proteins, urea denaturation experiments and DNA-binding studies were performed using fluorescence spectroscopy. The DNA-binding data showed similar dissociation constants for all proteins, with Kd values in the nanomolar range. The urea denaturation data, however, clearly indicated that the HZFB protein exhibited a completely different denaturation profile when compared to the ZFB, HG1b, and G1b proteins. The HZFB protein showed a profile indicative of the presence of an altered conformation around the sole tryptophan, whereas the other proteins showed a transition point between 3 and 4 M urea concentration. These data show that, although function was not altered for any of the proteins studied, the structure of one of the His-tagged proteins was different from the native form of that protein.
Chant
Alan
A
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK. achant@uvm.edu
Kraemer-Pecore
Christina M
CM
Watkin
Richard
R
Kneale
Geoff G
GG
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology; D016335:Zinc Fingers / Q000235:genetics*
D005656:Fungal Proteins; D006639:Histidine; D014364:Tryptophan; D014508:Urea; D013917:Thrombin; D003545:Cysteine