Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry.
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.
Sharma
Seema
S
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
Zheng
Haiyan
H
Huang
Yuanpeng J
YJ
Ertekin
Asli
A
Hamuro
Yoshitomo
Y
Rossi
Paolo
P
Tejero
Roberto
R
Acton
Thomas B
TB
Xiao
Rong
R
Jiang
Mei
M
Zhao
Li
L
Ma
Li-Chung
LC
Swapna
G V T
GV
Aramini
James M
JM
Montelione
Gaetano T
GT
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
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