Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement

Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn2+ mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.

[1]  T. Huber,et al.  Magic angle spinning NMR structure determination of proteins from pseudocontact shifts. , 2013, Journal of the American Chemical Society.

[2]  Robert G Griffin,et al.  Dynamic nuclear polarization at high magnetic fields. , 2008, The Journal of chemical physics.

[3]  Seymour H Konig.  A classical description of relaxation of interacting pairs of unlike spins: Extension to T1ϱ, T2, and T1ϱoff, including contact interactions , 1982 .

[4]  L. Brown,et al.  Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein , 2013, Nature Methods.

[5]  L. Brown,et al.  Paramagnetic relaxation enhancement reveals oligomerization interface of a membrane protein. , 2012, Journal of the American Chemical Society.

[6]  S. Opella,et al.  Structure determination of a membrane protein in proteoliposomes. , 2012, Journal of the American Chemical Society.

[7]  P. Bradley,et al.  Toward High-Resolution de Novo Structure Prediction for Small Proteins , 2005, Science.

[8]  Y. Ishii,et al.  Sensitivity enhancement, assignment, and distance measurement in 13C solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning. , 2006, Journal of magnetic resonance.

[9]  Jens Meiler,et al.  ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. , 2011, Methods in enzymology.

[10]  B. Meier,et al.  Distance information from proton-driven spin diffusion under MAS , 2006 .

[11]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[12]  Andrew E. Bennett,et al.  Heteronuclear decoupling in rotating solids , 1995 .

[13]  G. Wagner,et al.  Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. , 2000, Biochemistry.

[14]  Oliver F. Lange,et al.  NMR Structure Determination for Larger Proteins Using Backbone-Only Data , 2010, Science.

[15]  T. Ikegami,et al.  Utilization of paramagnetic relaxation enhancements for high-resolution NMR structure determination of a soluble loop-rich protein with sparse NOE distance restraints , 2014, Journal of Biomolecular NMR.

[16]  M. Eremets,et al.  Ammonia as a case study for the spontaneous ionization of a simple hydrogen-bonded compound , 2014, Nature Communications.

[17]  C. Jaroniec,et al.  Paramagnetic ions enable tuning of nuclear relaxation rates and provide long-range structural restraints in solid-state NMR of proteins. , 2009, Journal of the American Chemical Society.

[18]  Oliver F. Lange,et al.  Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples , 2012, Proceedings of the National Academy of Sciences.

[19]  S. Opella,et al.  Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) , 2014, Journal of Biomolecular NMR.

[20]  Alexander Pines,et al.  Proton‐enhanced NMR of dilute spins in solids , 1973 .

[21]  P. Güntert,et al.  Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR. , 2012, Structure.

[22]  David Baker,et al.  Resolution-adapted recombination of structural features significantly improves sampling in restraint-guided structure calculation , 2011, Proteins.

[23]  H. Oschkinat,et al.  Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain. , 2000, Journal of magnetic resonance.

[24]  R. Griffin,et al.  Proton assisted recoupling and protein structure determination. , 2008, The Journal of chemical physics.

[25]  B D Sykes,et al.  1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects , 1995, Journal of biomolecular NMR.

[26]  C. Jaroniec,et al.  Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of 15N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR , 2011, Journal of biomolecular NMR.

[27]  M. Engelhard,et al.  Secondary structure, dynamics, and topology of a seven-helix receptor in native membranes, studied by solid-state NMR spectroscopy. , 2007, Angewandte Chemie.

[28]  Andrew J. Nieuwkoop,et al.  Ultrahigh resolution protein structures using NMR chemical shift tensors , 2011, Proceedings of the National Academy of Sciences.

[29]  C. Jaroniec,et al.  Structural studies of proteins by paramagnetic solid-state NMR spectroscopy. , 2015, Journal of magnetic resonance.

[30]  Józef R. Lewandowski.  Advances in solid-state relaxation methodology for probing site-specific protein dynamics. , 2013, Accounts of chemical research.

[31]  R. Hodges,et al.  1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects , 1995, Journal of biomolecular NMR.

[32]  S. Opella,et al.  Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers , 2012, Nature.

[33]  Charles D Schwieters,et al.  The Xplor-NIH NMR molecular structure determination package. , 2003, Journal of magnetic resonance.

[34]  J. Bushweller,et al.  Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy. , 2006, Journal of the American Chemical Society.

[35]  Benjamin J. Wylie,et al.  Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. , 2005, Journal of the American Chemical Society.

[36]  M. Nilges,et al.  3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints. , 2008, Journal of the American Chemical Society.

[37]  D. Baker,et al.  High-resolution structure of a Shigella type III secretion needle by solid-state NMR and cryo-electron microscopy , 2014, Nature Communications.

[38]  D. Baker,et al.  Atomic model of the type III secretion system needle , 2012, Nature.

[39]  David Baker,et al.  Protein Structure Prediction Using Rosetta , 2004, Numerical Computer Methods, Part D.

[40]  Oliver F. Lange,et al.  Automatic NOESY assignment in CS-RASREC-Rosetta , 2014, Journal of Biomolecular NMR.

[41]  P. A. Kosen.  Spin labeling of proteins. , 1989, Methods in enzymology.

[42]  M. Baldus,et al.  Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. , 2002, Journal of the American Chemical Society.

[43]  N. Bloembergen.  Fine structure of the magnetic resonance line of protons in CuSO4.5H2O , 1950 .

[44]  Ivano Bertini,et al.  Solution NMR of Paramagnetic Molecules: Applications to metallobiomolecules and models , 2001 .

[45]  Soo-bin Park.  Hepatitis E vaccine debuts , 2012, Nature.

[46]  S. Wimperis,et al.  Solid-state NMR spectroscopy. , 2009, Physical chemistry chemical physics : PCCP.

[47]  B. Meier,et al.  De novo 3D structure determination from sub-milligram protein samples by solid-state 100 kHz MAS NMR spectroscopy. , 2014, Angewandte Chemie.

[48]  Kiyonori Takegoshi,et al.  13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR , 2001 .

[49]  Oliver F. Lange,et al.  Consistent blind protein structure generation from NMR chemical shift data , 2008, Proceedings of the National Academy of Sciences.

[50]  M. Schubert,et al.  Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy , 2002, Nature.

[51]  L. Brown,et al.  Solid-state NMR study of proteorhodopsin in the lipid environment: secondary structure and dynamics. , 2009, Biochimica et biophysica acta.

[52]  I. Solomon.  Relaxation Processes in a System of Two Spins , 1955 .

[53]  A. Bax,et al.  TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts , 2009, Journal of biomolecular NMR.

[54]  K. Zilm,et al.  Chemical shift referencing in MAS solid state NMR. , 2003, Journal of magnetic resonance.

[55]  Andrew J. Nieuwkoop,et al.  Supramolecular protein structure determination by site-specific long-range intermolecular solid state NMR spectroscopy. , 2010, Journal of the American Chemical Society.

[56]  B. Meier,et al.  Protein structure determination from 13C spin-diffusion solid-state NMR spectroscopy. , 2008, Journal of the American Chemical Society.

[57]  C. Schwieters,et al.  Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy , 2012, Nature chemistry.

[58]  K Wüthrich,et al.  The program XEASY for computer-supported NMR spectral analysis of biological macromolecules , 1995, Journal of biomolecular NMR.