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Abstract: We present here an improvisation of HNN (Panchal, Bhavesh et al., 2001) called RD 3D HNCAN for backbone (HN, CA and 15N) assignment in both folded and unfolded proteins. This is a reduced dimensionality experiment which employs CA chemical shifts to improve dispersion. Distinct positive and negative peak patterns of various triplet segments along the polypeptide chain observed in HNN are retained and these provide start and check points for the sequential walk. Because of co-incrementing of CA and 15N, peaks along one of the dimensions appear at sums and differences of the CA and 15N chemical shifts. This changes the backbone assignment protocol slightly and we present this in explicit detail. The performance of the experiment has been demonstrated using Ubiquitin and Plasmodium falciparum P2 proteins. The experiment is particularly valuable when two neighboring amino acid residues have nearly identical backbone 15N chemical shifts. [Copyright &y& Elsevier]

We describe here the tunability of the HNN experiment to obtain certain residue specific peak patterns in the spectra of (15N, 13C) labeled proteins. This is achieved by tuning a band-selective 180° pulse on the carbon channel in the pulse sequence, whereby one can tamper with the Cα–Cβ coupling evolutions for the different residues. Specifically, we generate distinctive peak patterns for serine and threonine and their neighbors in the different planes of the three dimensional spectrum. These provide useful anchor points during sequential assignment of backbone resonances. The performance of this experiment, referred to as HNN-ST here, is demonstrated using two proteins, one properly folded and the other completely denatured. With the availability of high field spectrometers, techniques such as TROSY, and ever increasing sensitivities in the probes, this experiment with its large number of check points has a great potential for rapid and unambiguous backbone resonance assignment in large proteins. [ABSTRACT FROM AUTHOR]