Measurement of Very Fast Exchange Rates of Individual Amide Protons in Proteins by NMR Spectroscopy.

NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 10  s . Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2N H by exchange events that occur during a series of pi pulses on the N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D , where exchange rates up to 600 s were measured for amides, where no signal was detectable in N- H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47-69 kJ mol for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.