Measurement of Amide15N–1H One-Bond Couplings in Proteins Using Accordion Heteronuclear-Shift-Correlation Experiments

The measurement of scalar coupling constants is assuming ful structural information can be derived from the coupling a role of increasing importance in structural and functional contribution (12).As these dipolar contributions are often studies of macromolecules.They can provide a valuable small, accurate measurement of the one-bond splitting under source of structural information that is complementary to consideration is essential.Furthermore, the scalar part of the distance constraints derived from the measurement of the coupling constant is likely to show small variations in 1 H-1 H NOEs and they can provide an indication of subtle response to local structure.Although these variations are not changes in electronic structure about specific pairs of couyet well documented for 15 N-1 H couplings, other one-bond pled nuclei.Furthermore, in isotope-enriched systems, a trescalar coupling constants have been demonstrated to be usemendous number of scalar couplings can be measured.There ful probes of local conformation.For example, both 1 J CaHa is tremendous incentive, therefore, for tackling the often and 1 J CaN couplings have been shown to correlate with backdifficult problem of accurately measuring these couplings bone f and c angles in proteins (9, 13).Furthermore, it has and then interpreting these measured couplings in terms of recently been reported that 1 J NC couplings are sensitive to useful structural and chemical models.We present here a H-bonding effects ( 14).series of steps toward improving accuracy in determiningIn this Note, we will focus on the optimization of methone-bond 15 N-1 H couplings from the indirect frequency doods in which the coupling of interest results in a splitting main of HSQC spectra.which is measured directly from the frequency domain of Experimentally, there is now a wide array of methods a transformed 2D spectrum.We will address utilization which have been designed to allow the accurate measureof resonance-intensity-based experiments in a subsequent ment of coupling constants (1).Among these methods, a paper.Frequency-domain experiments are not only connumber of fundamentally different approaches have been ceptually simple but are often less prone to systematic pursued.These include frequency-resolved methods, such as error than resonance-intensity-based experiments.On the J-resolved spectroscopy (2-4) and E. COSY experiments other hand, experiments of this type uniformly suffer from (5-7).They also include quantitative J-modulation experia degradation of precision as linewidths increase.Also,, in which the coupling is extracted from the they are time consuming as the data must be collected resonance intensity rather than by direct measurement of an with high digital resolution.When couplings are measured experimental splitting.The frequency-resolved versus intenin an indirect, f 1 , dimension with large chemical-shift dissity-based methods are somewhat complementary, as they persion, this requires a large number of t 1 points.J-reare often subject to different sources of sytematic error.They solved spectroscopy can be employed to improve precialso differ somewhat in applicability to different types of sion as field-inhomogeneity effects are refocused and the spin-coupling networks: two spin, three spin, and higher.spectral width which must be characterized in the indirect Our interest is in the measurement of one-bond coupling dimension is reduced to that required for just the spinconstants in proteins, in particular 1 J NH .In molecular sysspin couplings present.However, unless a third dimension tems which do not sample orientations isotropically during is employed, this compromises chemical-shift resolution tumbling in solution, measured one-bond splittings will conin the f 1 dimension ( 15 ) .Herein we propose a modificatain a dipolar coupling contribution.When deviations are tion of 2D J spectroscopy in which the indirect evolution due to magnetic field interactions, the magnitude is related period is actually a composite of two evolution periods to the orientation of the internuclear vector relative to the which are incremented accordion style ( 16 ) .A useful principal axes of the magnetic susceptibility tensor and usecompromise between spin -spin coupling and chemicalshift resolution can be achieved.Since the attainable precision will be limited by the