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

J Am Chem Soc. 2018 Mar 14;140(10):3518-3522. doi: 10.1021/jacs.8b00215. Epub 2018 Mar 6.

Abstract

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 (NH i-1, NH i) or flanking a proline at position i-1 (NH i-2, NH i). 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Amides / chemistry*
  • Humans
  • Nuclear Magnetic Resonance, Biomolecular*
  • Proline / chemistry*
  • alpha-Synuclein / chemistry*

Substances

  • Adaptor Proteins, Signal Transducing
  • Amides
  • B cell linker protein
  • alpha-Synuclein
  • Proline