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

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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 104 s−1. Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2NxHz by exchange events that occur during a series of pi pulses on the 15N 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 D9k, where exchange rates up to 600 s−1 were measured for amides, where no signal was detectable in 15N−1H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47–69 kJ mol−1 for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.

Original languageEnglish
Pages (from-to)231-235
Number of pages5
Publication statusPublished - 21 Jan 2019

    Research areas

  • hydrogen exchange, kinetics, NMR spectroscopy, protein folding, spin−spin decorrelation

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