Evaluation of Cross-Correlation Effects and Measurement of One-Bond Couplings in Proteins with Short Transverse Relaxation Times

Abstract

Various strategies are described and compared for measurement of one-bond JNH and JNC′ splittings in larger proteins. In order to evaluate the inherent resolution obtainable in the various experiments, relaxation rates of 15N–1HN coupled and heteronuclear decoupled resonances were measured at 600- and 800-MHz field strengths for both perdeuterated and protonated proteins. A comparison of decay rates for the two 15N–{HN} doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz 1H 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 15N 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 1H dimension, the upfield 1HN–{15N} component on average relaxes slower than the downfield 1HN–{15N} component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upfield 15N–{1H} doublet component in slowly tumbling proteins makes it advantageous to derive the JNH splitting from the difference in frequency between the narrow downfield 15N doublet component and either the 1H-decoupled 15N resonance or the peak position in an experiment which J-scales the frequency of the upfield doublet component but maintains some of the advantages of the TROSY experiment.