Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use 1 H β chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the 1 H β spins during the experiments is allowed to evolve for a fixed evolution period that results in phase modulation (positive or negative) of the cross peaks corresponding to various amino acid residues on their 2D HN projections, resembling a typical 2D [ 1 H- 15 N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their 1 H β chemical shifts, resulting in unique phase patterns at different fixed evolution periods for 1 H β , thus facilitating their identification. Remarkably, the PM-2D HN(HB) stands out among all amino acid type identification NMR techniques for its applicability with cost-effective and most routinely employed 15 N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the 13 C β chemical shift-based phase modulated NMR method (PM-2D HN(CACB)), these methods resolved the identification of large groups of amino acids into relatively smaller groups. Moreover, these techniques can accelerate the sequence-specific sequential resonance assignment (SSRA) process and would help in fast tracking of assigned NMR signals exhibiting chemical shift perturbation on the 2D [ 1 H- 15 N]-HSQC spectrum of proteins during various experiments (e.g., temperature change, pH change, and protein or ligand or cofactor binding) as well as in site-directed mutagenesis.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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