Unambiguous Tracking of Protein Phosphorylation by Fast High‐Resolution FOSY NMR**

Abstract

Dysregulation of post‐translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near‐physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3β) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

Specific phosphorylation of microtubule-associated protein 2c by extracellular signal–regulated kinase reduces interactions at its Pro-rich regions

“…Unambiguous assignment was obtained by recording 1 H, 15 N-HSQC spectra of four shorter MAP2c fragments (1–159, 159–254, 250–347, and 300–467) and of MAP2c mutants prepared by site-directed mutagenesis of serines and threonines followed by proline (mutants S28D, S136D, S140D, S178D, T245E, T259E, T262E, S264D, S274D, S422D, MAP2c-159–254/T238E, MAP2c-250–347/T268E, MAP2c-250–347/T271E, MAP2c-250–347/T289E, MAP2c-250–347/S293A). In the case of MAP2c phosphorylated by ERK2, assignment of Thr248 and unexpected phosphorylation at Ser274 were confirmed by following connectivities in 2D FOSY NMR spectra ( 44 ). The chemical shifts of the phosphorylated Ser/Thr are listed in Table S1 in Supplementary material.…”

“…2D hnco(CA)NH and hncoCA(N)H FOSY spectra ( 44 ) with selective excitation of the probed frequencies were acquired with spectral widths set to 18939 (aq) × 2500 ( 15 N) × 17921 ( 13 C′) Hz. The number of recorded complex points was 2048, 40, and 128 for 1 H, 15 N, and 13 C dimensions respectively.…”