Clean STD-NMR spectrum for improved detection of ligand-protein interactions at low concentration of protein
✍ Scribed by Youlin Xia; Qi Zhu; Kyu-Yeon Jun; Jingchun Wang; Xiaolian Gao
- Book ID
- 102532199
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 264 KB
- Volume
- 48
- Category
- Article
- ISSN
- 0749-1581
- DOI
- 10.1002/mrc.2687
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✦ Synopsis
Abstract
Saturation transfer difference (STD)‐NMR has been widely used to screen ligand compound libraries for their binding activities to proteins and to determine the binding epitopes of the ligands. We report herein, a Clean STD‐NMR method developed to overcome false positives (artifacts) observed in the STD‐NMR spectrum due to the power spillover of RF irradiation. The method achieved higher degree of resonance saturation through digital editing of two STD‐NMR spectra to generate a concatenated difference spectrum and three times of sensitivity enhancement for a loose binding complex involving DNA oligonucleotide and an RNA‐binding protein, CUGBP‐1ab (25.2 kDa). The interesting binding characteristics of the complex dCTGTCT–CUGBP1ab were obtained. The method was applied to a mixture of small ligand and bovine serum albumin protein (BSA, 66.3 kDa), and detected the intermolecular contacts at a BSA concentration as low as 0.1 µM, a working concentration useful for the detection of proteins of low solubility at biologically relevant conditions. Copyright © 2010 John Wiley & Sons, Ltd.