Accurate protein-peptide titration experiments by nuclear magnetic resonance using low-volume samples.
NMR spectroscopy allows measurements of very accurate values of equilibrium dissociation constants using chemical shift perturbation methods, provided that the concentrations of the binding partners are known with high precision and accuracy. The accuracy and precision of these experiments are improved if performed using individual capillary tubes, a method enabling full automation of the measurement. We provide here a protocol to set up and perform these experiments as well as a robust method to measure peptide concentrations using tryptophan as an internal standard.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1, rue Laurent Fries, BP 10142, 67404, Illkirch Cedex, France.
Christian
C
Köhler
Raphaël
R
Recht
Marc
M
Quinternet
Frederic
F
de Lamotte
Marc-André
MA
Delsuc
Bruno
B
Kieffer
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism; D000097768:Retinoic Acid Receptor gamma
D048868:Adaptor Proteins, Signal Transducing; D009124:Muscle Proteins; D010455:Peptides; D011506:Proteins; D018168:Receptors, Retinoic Acid; D000097768:Retinoic Acid Receptor gamma; C486523:SORBS3 protein, human