Dispersion from C(α) or N(H): 4D experiments for backbone resonance assignment of intrinsically disordered proteins

Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three H(α)-detected 4D experiments with C(α) as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D H(α)-start, H(N)-detect experiments which have two N(H) dimensions to enhance peak dispersion in a sequential walk through C′, N(H) and H(N), and provide more accurate N(H)/H(N) chemical shifts than those that can be obtained from a crowded (1)H, (15)N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10858-020-00299-w) contains supplementary material, which is available to authorized users.