Sensitivity-Enhanced Four-Dimensional Amide–Amide Correlation NMR Experiments for Sequential Assignment of Proline-Rich Disordered Proteins

Proline
is prevalent in intrinsically disordered proteins (IDPs).
NMR assignment of proline-rich IDPs is a challenge due to low dispersion
of chemical shifts. We propose here new sensitivity-enhanced 4D NMR
experiments that correlate two pairs of amide resonances that are
either consecutive (NHi–1, NHi) or flanking a proline at position i–1 (NHi–2, NHi). The maximum 2-fold enhancement of sensitivity
is achieved by employing two coherence order-selective (COS) transfers
incorporated unconventionally into the pulse sequence. Each COS transfer
confers an enhancement over amplitude-modulated transfer by a factor
of √2 specifically when transverse relaxation is slow. The
experiments connect amide resonances over a long fragment of sequence
interspersed with proline. When this method was applied to the proline-rich
region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein
(pH 7.4), which contain a total of 52 and 5 prolines, respectively,
99% and 92% of their nonprolyl amide resonances have been successfully
assigned, demonstrating its robustness to address the assignment problem
in large proline-rich IDPs.