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.
Original language | English (US) |
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Pages (from-to) | 918-924 |
Number of pages | 7 |
Journal | Magnetic Resonance in Chemistry |
Volume | 48 |
Issue number | 12 |
DOIs | |
State | Published - Dec 2010 |
Keywords
- Clean STD-NMR
- DNA-protein complex
- STD-NMR
- low concentration protein
- protein binding