Novel Techniques for Weak Alignment of Proteins in Solution Using Chemical Tags Coordinating Lanthanide Ions

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual 15N,1H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.

[1]  L. Kay,et al.  Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy. , 2001, Journal of the American Chemical Society.

[2]  J. Meiler,et al.  Dipolar couplings in multiple alignments suggest alpha helical motion in ubiquitin. , 2003, Journal of the American Chemical Society.

[3]  Che Ma,et al.  Lanthanide ions bind specifically to an added "EF-hand" and orient a membrane protein in micelles for solution NMR spectroscopy. , 2000, Journal of magnetic resonance.

[4]  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.

[5]  Rafael Brüschweiler,et al.  Self-consistency analysis of dipolar couplings in multiple alignments of ubiquitin. , 2003, Journal of the American Chemical Society.

[6]  S. Block,et al.  Mixed thiolsulfonates and sulfonamides from polyfunctional mercaptans using trifluoromethyl thiosulfonates. , 1972, Journal of medicinal chemistry.

[7]  C. Meares,et al.  Protein-protein interactions mapped by artificial proteases: where sigma factors bind to RNA polymerase. , 2000, Trends in biochemical sciences.

[8]  I. Bertini,et al.  Paramagnetism-based versus classical constraints: An analysis of the solution structure of Ca Ln calbindin D9k , 2001, Journal of biomolecular NMR.

[9]  R. Ebright,et al.  Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro. , 1992, Biochemistry.

[10]  J Meiler,et al.  Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. , 2001, Journal of the American Chemical Society.

[11]  M. D. Kemple,et al.  The use of lanthanides for solution structure determination of biomolecules by NMR. Evaluation of the methodology with EDTA derivatives as model systems , 1988 .

[12]  K. Fiebig,et al.  NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein. , 2002, Journal of molecular biology.

[13]  A. Rosato,et al.  Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix. , 2001, Journal of the American Chemical Society.

[14]  J. R. Tolman.  A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy. , 2002, Journal of the American Chemical Society.

[15]  J. M. Delfino,et al.  Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[18]  G. N. Mar,et al.  Proton spin relaxation for the nonlabile coordinated chelate in lanthanide shift reagents , 1982 .

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

[20]  M. Gochin.  A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance. , 2000, Structure.

[21]  S. Grzesiek,et al.  Purple membrane induced alignment of biological macromolecules in the magnetic field , 1999 .

[22]  M. Pons,et al.  Measurement of One Bond Dipolar Couplings through Lanthanide-Induced Orientation of a Calcium-Binding Protein , 1999 .

[23]  A M Gronenborn,et al.  New methods of structure refinement for macromolecular structure determination by NMR. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  V. Volkov,et al.  Novel chelate-induced magnetic alignment of biological membranes. , 1998, Biophysical journal.

[25]  B. Volkman,et al.  Determination of internuclear angles of DNA using paramagnetic-assisted magnetic alignment. , 1998, Journal of magnetic resonance.

[26]  Joel R Tolman,et al.  De novo determination of bond orientations and order parameters from residual dipolar couplings with high accuracy. , 2003, Journal of the American Chemical Society.

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

[28]  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.

[29]  Harald Schwalbe,et al.  Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings. , 2003, Journal of the American Chemical Society.

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

[31]  Jens Meiler,et al.  Model-free analysis of protein backbone motion from residual dipolar couplings. , 2002, Journal of the American Chemical Society.

[32]  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.

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