Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Seema Sharma, Haiyan Zheng, Yuanpeng J. Huang, Asli Ertekin, Yoshitomo Hamuro, Paolo Rossi, Roberto Tejero, Thomas B. Acton, Rong Xiao, Mei Jiang, Li Zhao, Li Chung Ma, G. V.T. Swapna, James M. Aramini, Gaetano T. Montelione

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three-dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of 1H/2H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N- and C-terminal tails were evaluated using 1H-15N HSQC and 1H-15N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS-based truncated construct for a 77-residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination.

Original languageEnglish (US)
Pages (from-to)882-894
Number of pages13
JournalProteins: Structure, Function and Bioinformatics
Volume76
Issue number4
DOIs
StatePublished - Sep 2009

Keywords

  • DXMS
  • Hydrogen-deuterium exchange
  • Mass spectrometry
  • NMR
  • Partially disordered proteins
  • Protein construct optimization
  • Structural genomics

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