Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

We demonstrate improved accuracy in protein structure determination for large (≥30 kDa), deuterated proteins (e.g. STAT4NT) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.

[1]  P. Kraulis,et al.  Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing. , 1989, Biochemistry.

[2]  G. Montelione,et al.  A general approach for determining scalar coupling constants in polypeptides and proteins , 1992, Biopolymers.

[3]  P. Kraulis,et al.  Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy. , 1994, Biochemistry.

[4]  B. Farmer,et al.  High-level 2H/13C/15N labeling of proteins for NMR studies , 1995, Journal of biomolecular NMR.

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

[6]  J H Prestegard,et al.  Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[8]  L. Kay,et al.  An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins , 1996, Journal of biomolecular NMR.

[9]  L. Kay,et al.  Solution NMR spectroscopy beyond 25 kDa. , 1997, Current opinion in structural biology.

[10]  L. Kay,et al.  Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR. , 1997, Biochemistry.

[11]  R. Riek,et al.  Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Bax,et al.  Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. , 1997, Science.

[13]  J. Darnell,et al.  Structure of the amino-terminal protein interaction domain of STAT-4. , 1998, Science.

[14]  Ad Bax,et al.  Modulation of the Alignment Tensor of Macromolecules Dissolved in a Dilute Liquid Crystalline Medium , 1998 .

[15]  L. Kay,et al.  An NMR Experiment for Measuring Methyl−Methyl NOEs in 13C-Labeled Proteins with High Resolution , 1998 .

[16]  M. Gochin.  Nuclear Magnetic Resonance Characterization of a Paramagnetic DNA-drug Complex with High Spin Cobalt; Assignment of the 1H and 31P NMR Spectra, and Determination of Electronic, Spectroscopic and Molecular Properties , 1998, Journal of biomolecular NMR.

[17]  L. Kay,et al.  A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins , 1999, Journal of biomolecular NMR.

[18]  A. Bax,et al.  Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values , 1999, Journal of biomolecular NMR.

[19]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[20]  J. Hus,et al.  De novo determination of protein structure by NMR using orientational and long-range order restraints. , 2000, Journal of molecular biology.

[21]  L. Kay,et al.  Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein. , 2000, Journal of molecular biology.

[22]  I. Bertini,et al.  Lanthanide-Induced Pseudocontact Shifts for Solution Structure Refinements of Macromolecules in Shells up to 40 Å from the Metal Ion , 2000 .

[23]  G M Clore,et al.  Direct refinement against proton-proton dipolar couplings in NMR structure determination of macromolecules. , 2000, Journal of magnetic resonance.

[24]  P. Rosevear,et al.  Calculation of z-coordinates and orientational restraints using a metal binding tag. , 2000, Biochemistry.

[25]  J. Hus,et al.  Determination of protein backbone structure using only residual dipolar couplings. , 2001, Journal of the American Chemical Society.

[26]  P. Bayley,et al.  Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution , 2001, Journal of biomolecular NMR.

[27]  L. Kay,et al.  Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein , 2001, Journal of biomolecular NMR.

[28]  L. Kay,et al.  What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR. , 2001, Journal of molecular biology.

[29]  I. Bertini,et al.  Paramagnetic probes in metalloproteins. , 2001, Methods in enzymology.

[30]  L. Kay,et al.  Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. , 2002, Journal of the American Chemical Society.

[31]  D. Baker,et al.  De novo determination of protein backbone structure from residual dipolar couplings using Rosetta. , 2002, Journal of the American Chemical Society.

[32]  P. Rosevear,et al.  Derivation of structural restraints using a thiol‐reactive chelator , 2002, FEBS letters.

[33]  V. Gaponenko,et al.  Breaking symmetry in the structure determination of (large) symmetric protein dimers , 2002, Journal of biomolecular NMR.

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