Practical methods for solid-state NMR distance measurements on large biomolecules: Constant-time rotational resonance

Simple modifications of the rotational resonance experiment substantially r educe the total experimental time needed to measure weak homonuclear dipola r couplings, a critical factor for achieving routine internuclear distance measurements in large biomolecular systems. These modifications also addres s several problems cited in the literature. Here we introduce a constant-ti me rotational resonance experiment that eliminates the need for control spe ctra to correct for effects from variable RF heating, particularly critical for accurate long-distance measurements. This reduces the total number of experiments needed by as much as a factor of 2. Other improvements incorpor ated include achieving selective inversion with a delay rather than a weak pulse (P. R. Costa et al., J. Am. Chem. Sec. 119, 10487-10493, 1997), which we observe results in the elimination of oscillations in peak intensities for short mixing time points. This reduces the total experiment time in two ways. First, there is no longer a need to average different "zero"-time po ints (S. O. Smith et at, Biochemistry 33, 6334-6341, 1994) to correct for i ntensity variations. Second, short-mixing-time lineshape differences observ ed in large membrane-bound proteins only appear with the weak-pulse inversi on and not when using the delay inversion. Consistent lineshapes between sh ort and long mixing times permit the use of a single spectrum for subtracti on of natural abundance background signals from all labeled-protein time po ints. Elimination of these effects improves the accuracy and efficiency of rotational resonance internuclear distance measurements. (C) 1999 Academic Press.