TY - JOUR
T1 - Assignment strategies for large proteins by magic-angle spinning NMR
T2 - The 21-kDa disulfide-bond-forming enzyme DsbA
AU - Sperling, Lindsay J.
AU - Berthold, Deborah A.
AU - Sasser, Terry L.
AU - Jeisy-Scott, Victoria
AU - Rienstra, Chad M.
N1 - Funding Information:
The authors thank the National Institutes of Health for funding this work through the National Institute of General Medical Sciences and Roadmap Initiative ( GM075937 ) and a Molecular Biophysics Training grant ( PHS 5 T32 GM008276 ) to L.J.S. The authors also thank Drs. Ying Li, Trent Franks (School of Chemical Sciences NMR Facility at the University of Illinois at Urbana-Champaign), Heather Frericks-Schmidt, and Benjamin Wylie for assistance with data acquisition and helpful discussions, and Profs. Kenji Inaba (Kyushu University) and Koreaki Ito (Kyoto University) for their gift of expression plasmids for DsbA and DsbB.
PY - 2010/6/4
Y1 - 2010/6/4
N2 - We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to ~17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-13C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-13C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional 13C-13C spectrum.
AB - We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to ~17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-13C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-13C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional 13C-13C spectrum.
KW - Chemical shift assignment
KW - Correlation spectroscopy
KW - Dipolar recoupling
KW - Selective pulses
KW - Solid-state NMR
UR - http://www.scopus.com/inward/record.url?scp=77953082958&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77953082958&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2010.04.012
DO - 10.1016/j.jmb.2010.04.012
M3 - Article
C2 - 20394752
AN - SCOPUS:77953082958
VL - 399
SP - 268
EP - 282
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -