Accurate protein-peptide titration experiments by nuclear magnetic resonance using low-volume samples

Christian Köhler; Raphaël Recht; Marc Quinternet; Frederic de Lamotte; Marc-André Delsuc; Bruno Kieffer

doi:10.1007/978-1-4939-2447-9_22

Accurate protein-peptide titration experiments by nuclear magnetic resonance using low-volume samples

Methods Mol Biol. 2015:1286:279-96. doi: 10.1007/978-1-4939-2447-9_22.

Authors

Christian Köhler¹, Raphaël Recht, Marc Quinternet, Frederic de Lamotte, Marc-André Delsuc, Bruno Kieffer

Affiliation

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1, rue Laurent Fries, BP 10142, 67404, Illkirch Cedex, France.

PMID: 25749962
DOI: 10.1007/978-1-4939-2447-9_22

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / chemistry
Adaptor Proteins, Signal Transducing / metabolism
Amino Acid Sequence
Humans
Magnetic Resonance Spectroscopy / methods*
Models, Molecular
Molecular Sequence Data
Muscle Proteins / chemistry
Muscle Proteins / metabolism
Peptides / chemistry*
Peptides / metabolism*
Phosphorylation
Protein Structure, Tertiary
Proteins / chemistry*
Proteins / metabolism*
Receptors, Retinoic Acid / chemistry
Receptors, Retinoic Acid / metabolism
Retinoic Acid Receptor gamma

Substances

Adaptor Proteins, Signal Transducing
Muscle Proteins
Peptides
Proteins
Receptors, Retinoic Acid
SORBS3 protein, human