A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study

the c-terminal domain of histone h1.0 (c-h1.0) is involved in dna binding and is a main determinant of the chromatin condensing properties of histone h1.0. phosphorylation at the (s/t)-p-x-(k/r) motifs affects dna binding and is crucial for regulation of c-h1.0 function. since c-h1.0 is an intrinsically disordered domain, solution nmr is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of c-h1.0. however, its very repetitive, low-amino acid-diverse and pro-rich sequence, together with the low signal dispersion observed at the 1h–15n hsqc spectra of both non- and tri-phosphorylated c-h1.0 preclude the use of standard 1h-detected assignment strategies. we have achieved an essentially complete assignment of the heavy backbone atoms (15n, 13c′ and 13cα), as well as 1hn and 13cβ nuclei, of non- and tri-phosphorylated c-h1.0 by applying a novel 13c-detected con-based strategy. no c-h1.0 region with a clear secondary structure tendency was detected by chemical shift analyses, confirming at residue level that c-h1.0 is disordered in aqueous solution. phosphorylation only affected the chemical shifts of phosphorylated thr’s, and their adjacent residues. heteronuclear {1h}–15n noes were also essentially equal in the non- and tri-phosphorylated states. hence, structural tendencies and dynamic properties of c-h1.0 free in aqueous solution are unmodified by phosphorylation. we propose that the assignment strategy used for c-h1.0, which is based on the acquisition of only a few 3d spectra, is an excellent choice for short-lived intrinsically disordered proteins with repetitive sequences.