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

J Magn Reson. 2000 Mar;143(1):184-96. doi: 10.1006/jmre.1999.1979.

Abstract

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 (15)N-(1)H(N) 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 (15)N-¿H(N)¿ doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz (1)H 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 (15)N 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 (1)H dimension, the upfield (1)H(N)-¿(15)N¿ component on average relaxes slower than the downfield (1)H(N)-¿(15)N¿ component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upfield (15)N-¿(1)H¿ doublet component in slowly tumbling proteins makes it advantageous to derive the J(NH) splitting from the difference in frequency between the narrow downfield (15)N doublet component and either the (1)H-decoupled (15)N 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Magnetic Resonance Spectroscopy / methods*
  • Proteins / chemistry

Substances

  • Proteins