Novel relaxation compensated method to measure proton exchange rates in biomolecules based on decorrelation of heteronuclear two-spin order

Abstract

A combined experiment based on decorrelation of heteronuclear two-spin order and a new analysis method is presented for measuring accurate rapid amide proton exchange rates (kexHH = kex) in 15N-labeled biomolecules, such as proteins or nucleic acids, in water. The term ‘decorrelation’ is defined as the loss of the initial correlation between a labile biomolecule proton and its coupled nitrogen when they are separated by intermolecular chemical exchange with water. The NMR pulse sequence [DECORrelated EXchange SpectroscopY (DECOREXSY)] measures the decay of the heteronuclear two-spin order terms with minimal interference effects from relaxation processes and solvent-induced artifacts. The new analysis protocol based on backbone relaxation measurements is introduced to compensate for relaxation contributions to the exchange rates that are otherwise inseparable. This simple and straightforward scheme has several potential applications in protein folding and biomolecular recognition and binding studies, and fills the need for a sensitive experiment to measure absolute fast-amide proton exchange rates predominantly on the sub-millisecond time-scale. Copyright © 2000 John Wiley & Sons, Ltd.