Practical Aspects of Carbon-13 Double Quantum NMR
We want to address an erroneous view that has recently been advanced on 13C double quantum NMR. A recent publication (I) introduced a modified version (I) of the original double quantum NMR experiments; this new version claims to offer an attractive increase in sensitivity by a factor of three, or a decrease in the size of the required data matrix by a factor of four. To avoid disappointment among spectroscopists we wish to make it clear that this modified version offers no gain in sensitivity whatsoever in comparison with the other double quantum experiments (2-6), provided they are properly optimized. It is shown here that the sensitivity of this modified experiment can be even slightly lower. The claim of a reduction of the size of the required data matrix is also shown to be incorrect. In the original two-dimensional double quantum “C experiments, the acquisition time t2 starts immediately after the final “read pulse” (Fig. 1), and the time during which the double quantum coherence evolves is labeled ti. The detected 13C-13C satellites (7) are modulated in amplitude as a function of tl with the double quantum frequencies (2). In the quadrature-Fi-detection version of this experiment (3), results are combined with those of a second experiment which contains a composite 45: pulse (8) in the evolution period. This allows the sign of the modulation frequency to be determined, which simplifies the interpretation of the results. A simpler and more sensitive alternative to obtain quadrature F, detection has been proposed more recently (6); an increase of the flip angle of the final “read pulse” to 135” provides directly satellite signals which are modulated in phase as a function oft,. In either case, data acquisition is started after the final read pulse and the coherence transfer echo (9) is detected (4, 6). The time domain signal of a carbon A, directly coupled to carbon X, is then given by sA(tl, t2) = c exp[-i(nA + &)t,] exp[i& + d&2]
with a Large Double-Quantum Spectral Width
d it refl For p m, c bons, w er of t onyl c s ayed a t nect obt w ply m SI eq pling p s can s , MR spectra with a large spectral window in the double-quantum imension is presented. Using the dipolar recoupling sequence C7 o excite the double-quantum coherence under magic-angle spining, the technique involves incrementing the evolution period in ynchrony with the phase of the radiofrequency pulses in the C7 equence. The technique is demonstrated on a uniformly Cabeled amino acid and an extensively C-labeled protein to dentify C connectivity patterns for spectral assignment. © 1999
A 1H nuclear-magnetic-resonance study of the conformation and the molecular dynamics of the glycoprotein cow-colostrum trypsin inhibitor.
The glycoprotein cow colostrum trypsin inhibitor was investigated by high resolution 1H nuclear magnetic resonance (NMR) at 360 MHz and, on the basis of the NMR data, compared with the basic pancreatic trypsin inhibitor (Kunitz) from bovine tissue. Detailed studies of the chemical shifts and the exchange kinetics of the labile protons indicated extensive homologies between the spatial structures of the polypeptide chains in the two compounds. This was further corroborated by comparison of the NMR spectral features and the dynamic properties of the aromatic amino acid residues in the two inhibitors. It thus appears that the covalently attached carbohydrate moiety in the colostrum inhibitor has only very limited effects on the spatial structure of the protein part of the molecule. On the other hand, as evidenced by the NMR line widths, the carbohydrate attachment causes a pronounced restriction of the overall mobility of the molecule, indicating a sizeable increase of the average radius of gyration as compared to the basic pancreatic trypsin inhibitor. Possible spatial arrangements of the globular polypeptide and the carbohydrate moieties in the colostrum inhibitor, which would be compatible with the experimental observations, are discussed.