Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times

Various strategies are described and compared for measurement of one-bond J(NH) and J(NC), splittings in larger proteins. In order to evaluate the inherent resolution obtainable in the various experiments, relaxation rates of N-15-H-1(N) coupled and heteronuclear decoupled resonances were measured at 600- and 800-MHz held strengths for both perdeuterated and protonated proteins. A comparison of decay rates for the two N-15-{H-N} doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz H-1 frequency, respectively, in the perdeuterated proteins, For the protonated proteins these ratios are 3.2 (800 MHz) and 2.4 (600 MHz). Relative to the regular HSQC experiment, the enhancement in TROSY N-15 resolution is 2.6 (perdeuterated; 800 MHz), 2.0 (perdeuterated; 600 MHz), 2.1 (protonated; 800 MHz), and 1.7 (protonated; 600 MHz). For the H-1 dimension, the upheld H-1(N)-{N-15} component on average relaxes slower than the downfield H-1(N)-{N-15} component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upheld N-15-{H-1} doublet component in slowly tumbling proteins makes it advantageous to derive the J(NH) Splitting from the difference in frequency between the narrow downfield N-15 doublet component and either the H-1-decoupled N-15 resonance or the peak position in an experiment which J-scales the frequency of the upheld doublet component but maintains some of the advantages of the TROSY experiment.