Unambiguous tracking of protein phosphorylation by fast, high-resolution FOSY NMR

TL;DR

This paper introduces a fast, high-resolution NMR method called FOSY for unambiguously tracking protein phosphorylation sites, especially in large disordered proteins, improving efficiency and specificity over traditional approaches.

Contribution

The authors develop a novel FOSY NMR technique utilizing selective polarisation transfer to rapidly assign relevant signals, enabling precise identification of phosphorylation sites in complex proteins.

Findings

Successfully identified two phosphorylation sites in GSK3β on human Tau40.

Discovered GSK3β can phosphorylate Ser409 without priming, a novel finding.

Demonstrated FOSY's efficiency and robustness in studying PTMs in large IDPs.

Abstract

Phosphorylation is a prototypical example of post-translational modifications (PTMs) that dynamically modulate protein func-tion, where dysregulation is often implicated in disease. NMR provides information on the exact location and time course of PTMs with atomic resolution and under nearly physiological conditions, including inside living cells, but requires unambiguous prior assignment of affected NMR signals to individual atoms. Yet, existing methods for this task base on a global, hence, costly and tedious NMR signal assignment that may often fail, especially for large intrinsically disordered proteins (IDPs). Here we introduce a sensitive and robust method to rapidly obtain only the relevant local NMR signal assignment, based on a suite of FOcused SpectroscopY (FOSY) experiments that employ the long overlooked concept of selective polarisation transfer (SPT). We then demonstrate the efficiency of FOSY in identifying two phosphorylation sites of proline-dependent glycogen synthase kinase 3 beta (GSK3\b{eta}) in human Tau40, an IDP of 441 residues. Besides confirming the known target residue Ser404, the un-precedented spectral dispersion in FOSY disclosed for the first time that GSK3\b{eta} can also phosphorylate Ser409 without priming by other protein kinases. The new approach will benefit NMR studies of other PTMs and protein hotspots in general, including sites involved in molecular interactions and conformational changes