FOSY: Segmental Backbone Assignment in Intrinsically Disordered Proteins

RESUMO

Backbone resonance assignment is a prerequisite for most biomolecular NMR applications, yet conventional multidimensional strategies frequently fail for intrinsically disordered proteins (IDPs) and regions (IDRs) because of severe spectral overlap, rapid amide proton exchange with water, and missing sequential correlations. In many biological applications, however, complete protein assignment is unnecessary, as only a limited sequence segment surrounding a functional site is required. Here we introduce segmental backbone assignment, an assignment strategy implemented by FOcused SpectroscopY (FOSY), which concentrates experimental effort on relatively short regions while retaining the high-dimensional sequential connectivity needed for unambiguous assignments. We present a self-consistent suite of selective two-dimensional FOSY experiments that enables bidirectional assignment walks along the protein sequence through complementary forward and backward transfer schemes. The methodology employs frequency-selective polarisation transfer to replace high-dimensional experiments with sensitive and readily interpretable 2D spectra while preserving the information content of multidimensional correlation experiments. The approach is demonstrated by completing the assignment of G302-K311 segment, which is missing in the published assignment of the 441-residue human Tau protein. The approach complements conventional multidimensional or residue type-selective assignment strategies by providing an efficient means of traversing assignment interruptions and rapidly characterising functionally important segments in intrinsically disordered proteins.