title: Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry.
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 (1)H/(2)H 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 (1)H-(15)N HSQC and (1)H-(15)N 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.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Sharma
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zheng
authors.forename: Yuanpeng J
authors.initials: YJ
authors.lastname: Huang
authors.forename: Asli
authors.initials: A
authors.lastname: Ertekin
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.forename: Roberto
authors.initials: R
authors.lastname: Tejero
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Mei
authors.initials: M
authors.lastname: Jiang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Li-Chung
authors.initials: LC
authors.lastname: Ma
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: James M
authors.initials: JM
authors.lastname: Aramini
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000737:chemistry; D017173:Caenorhabditis elegans / Q000737:chemistry; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000191:economics / Q000379:methods*; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013997:Time Factors
chemical_list: D000577:Amides; D029742:Caenorhabditis elegans Proteins; D029968:Escherichia coli Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; C544711:YaiD protein, E coli; C544701:tppp-1 protein, C elegans