H chemical shift-based phase modulated NMR methods for fast identification of amino acid types in proteins.

Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use H chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the H 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 [H-N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their H chemical shifts, resulting in unique phase patterns at different fixed evolution periods for H, 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 N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the C 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 [H-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.