Exchange Rate Constants of Invisible Protons in Proteins Determined by NMR Spectroscopy
Although labile protons that are exchanging rapidly with those of the solvent cannot be observed directly, their exchange rate constants can be determined by indirect detection of scalar-coupled neighboring nuclei. We have used heteronuclear NMR spectroscopy to measure the exchange rate constants of labile protons in the side chains of lysine and arginine residues in ubiquitin enriched in carbon-13 and nitrogen-15 at neutral pH. Exchange rate constants as fast as 40×103 s-1 were thus measured. These results demonstrate that NMR spectroscopy is a powerful tool for the characterization of lysine NH3+ and arginine NH groups in proteins at physiologically relevant pH values.