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

A combined experiment based on decorrelation of heteronuclear two-spin orde r and a new analysis method is presented for measuring accurate rapid amide proton exchange rates (k(ex)(HH) = k(ex)) in N-15-labeled biomolecules, su ch as proteins or nucleic acids, in water. The term 'decorrelation' is defi ned as the loss of the initial correlation between a labile biomolecule pro ton and its coupled nitrogen when they are separated by intermolecular chem ical exchange with water. The NMR pulse sequence [DECORrelated EXchange Spe ctroscropY (DECOREXSY)] measures the decay of the heteronuclear two-spin or der terms with minimal interference effects from relaxation processes and s olvent-induced artifacts. The new analysis protocol based on backbone relax ation measurements is introduced to compensate for relaxation contributions to the exchange rates that are otherwise inseparable. This simple and stra ightforward scheme has several potential applications in protein folding an d biomolecular recognition and binding studies, and fills the need for a se nsitive experiment to measure absolute fast-amide proton exchange rates pre dominantly on the sub-millisecond time-scale. Copyright (C) 2000 John Wiley & Sons, Ltd.