Direct cross-relaxation NOESY (D.NOESY). A method for removing spin-diffusion cross peaks from two-dimensional NOE spectra of macromolecules

Abstract

Spin-diffusion effects compromise the interpretation of nuclear Overhauser effects in 1 D ( 1, 2) and 2D (3-5) spectroscopy. Problems arise when multistep (two or more) magnetization exchange occurs during the mixing time. The geometrical arrangement of protons in macromolecules provides many such pathways, which lead (i) to the appearance of multistep cross-relaxation cross peaks from pairs of spins that are not near neighbors and (ii) to the reduction of volumes of the direct cross-relaxation peaks. A further complication arises when cross relaxation is mediated by local mobility of parts of the molecule; such effects, although important ( 6)) are not addressed here. Spin-diffusion effects can be particularly pronounced when geminal protons are involved in the pathway. Geminal protons ( j, k), which are separated by a short fixed distance (about 1.8 A), are found to exhibit cross-relaxation rates from 2 to 100 times faster than those of other pairs of protons (k, I) located in the same molecule but separated by 2-4 A (7 ) . Consider a linear chain (j . * k. . . I) with direct cross-relaxation rates uk( and aj/. Misinterpretation of two-step spin diffusion ( ajk * uki) as direct cross relaxation u,/ may lead to underestimation of the distance r,/ by as much as 30% (when (Tk/’ 7, = 0.1 and ffjk/ok/ = 100). With longer mixing times, magnetization transfer may involve additional steps and lead to an even larger underestimation of the distance. One normally attempts to include distances derived from as many cross peaks as possible as input data for algorithms used in structure calculations (e.g., distance geometry). This is because a larger number of distance constraints leads to a tighter convergence of the calculated structures. The problem is that many of the weaker peaks do not represent direct cross relaxation and some direct peaks may have perturbed intensities. The danger is that the erroneous distances, derived from these cross peaks, will contribute to a set of convergent three-dimensional structures which, although consistent with the input distances, would not model the molecule accurately.