HNCA experiments

Fig. 4.1 Schematic diagram of the magnetization transfer pathway in the HNCA experiment. Two pathways exist, that transfer the magnetization from the amide proton to the amide nitrogen and then either to the intra-residual a-carbon (black...

Fig. 4.2 Strips taken from an HNCA experiment of the transcription factor CDC5. The amide proton frequency is along the x-axis, the 13Ca frequency is along the y-axis and the 15N-frequency is perpendicular to the planes shown. Intra-...

X/Y coherence transfer steps as in the original HNCA experiment.41 As in the previous case, X nuclei are selectively irradiated by low power rectangular or shaped pulses, and coherence selection is accomplished by the matched pulsed field gradients Gb G2 and further assisted by a spoil gradient Gs. Owing to the need to avoid -pulses in the X-channel, the spectra are processed in magnitude mode in the fft dimension. 

An HNCA experiment designed for 2H,i5N,13C labelled proteins was recorded as described by Yamazaki et al (19). This experiment was acquired on a three channel Varian UnitySOO spectrometer modified to perform the i N pulses, i5N decoupling and 2H decoupling on a single channel. Alternatively, this could be accomplisted on a four channel instrument without modification. 

Heteronuclear Triple Resonance Experiments. - A very small number of new triple-resonance experiments was reported. A version of the 3D HNCA experiment was presented that suppresses coherence transfer through /(N,C ). The method is based on the scalar couplings of sequential and to the same which is not the case for the intraresidue and... 

H, C, Si, and using two cardinal types of pulse sequences based on the HNCO and HNCA experiments which constitute standard schemes routinely employed for structure elucidation of peptides and proteins. However, whereas work on biomolecules is generally carried out with uniformly and labelled molecules, these studies were performed in natural abundance of isotopes, which required some adaptations of the experiments in order to improve the suppression of signals from isotopomers with magnetically inactive and Si nuclei. 

The HNCA experiment has been described in detail already and need not be discussed much further here (see Section 5.4.2), except to note that the HNCA experiment is a tripleresonance experiment in that it generates intensity data Inmr(Fi, Fj, F3) emanating from the triple resonance of three entirely different populations of and nuclei. 

The workings of the 3D HNCA experiment are illustrated diagrammatically in Figure 5.24. Frequently, HNCA spectral data is displayed in the form of individual C-, frequency (F2 and F3) contour maps resolved at different frequencies (H) (all in ppm). However, data could equally well be plotted in the form of H-frequency (Fi and F3) contour maps if this were more helpful for assignment purposes. Usually the HNCA experiment is combined with other 3D correlation experiments with names such as HNCO, HCACO and... 

HCA(CO)N experiments. These names are deliberately descriptive and should suggest the correlations that these experiments are intended to establish by analogy with the HNCA experiment described here. All these correlation experiments acting in concert are designed to ensure optimal and mostly complete, unique and unambiguous assignments of Tf-resonance peaks to nuclei in given protein NMR spectra. This level of information is usually sufficient... 

---