Clean STD-NMR spectrum for improved detection of ligand-protein interactions at low concentration of protein.
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.
Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA. yxia@uh.edu
Youlin
Y
Xia
Qi
Q
Zhu
Kyu-Yeon
KY
Jun
Jingchun
J
Wang
Xiaolian
X
Gao
D000818:Animals; D002417:Cattle; D008024:Ligands; D057230:Limit of Detection; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D009841:Oligonucleotides; D011506:Proteins; D012710:Serum Albumin, Bovine