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

Ikegami, Takahisa; Verdier, Laurent; Sakhaii, Peyman; Grimme, Susanne; Pescatore, Barbara; Saxena, Krishna; Fiebig, Klaus M.; Griesinger, Christian

doi:10.1023/b:jnmr.0000032611.72827.de

Cited by 134 publications

(169 citation statements)

References 33 publications

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“…However, the magnetic susceptibility anisotropy of cobalt(II) in the MMP-1 CAT domain is not large enough to provide measurable effects as far away as needed to reach the HPX domain nuclei. The introduction of covalently bound lanthanide chelators has been widely exploited to introduce paramagnetic centers in diamagnetic proteins (47)(48)(49); the rigid lanthanide chelator CLaNP-5 (50) does so by covalently binding two neighboring Cys residues in a rigid fashion. CLaNP-5 was incorporated into the MMP-1 CAT domain via Cys 132 and Cys 136 .…”

Section: Resultsmentioning

confidence: 99%

Examination of Matrix Metalloproteinase-1 in Solution

Cerofolini¹,

Fields

Fragai

et al. 2013

Journal of Biological Chemistry

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Background: Matrix metalloproteinase-1 (MMP-1) collagenolysis relies on interdomain flexibility. Results: In all high maximum occurrence conformations, the MMP-1 hemopexin-like domain residues reported responsible for binding to the collagen triple-helix are solvent exposed. Conclusion: MMP-1 in solution is poised to interact with collagen and proceed along the steps of collagenolysis. Significance: The maximum occurrence approach can evaluate the predominant domain conformations for numerous multidomain enzymes.

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Section: Resultsmentioning

confidence: 99%

Examination of Matrix Metalloproteinase-1 in Solution

Cerofolini¹,

Fields

Fragai

et al. 2013

Journal of Biological Chemistry

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“…It is worth recalling that paramagnetic metals, besides being incorporated in metalloproteins in the place of diamagnetic ones, can be also attached as tags to any protein (43,44,49), and this renders the approach quite general. All of the samples were crystallized by following the reported procedure (38,42), with minor changes: By using aliquots of 50 l of 1.5 mM protein solution [10 mM Tris/5 mM CaCl2/1 mM CoCl2/300 mM NaCl/3 mM NNGH (pH 7.0)] mixed with 50 l of reservoir buffer [0.1 M Tris⅐HCl/25% PEG 8000 (pH 8.…”

Section: Discussionmentioning

confidence: 99%

“…The present approach is general and independent of the information coming from X-ray diffraction techniques. Even if the present method is demonstrated on a metalloprotein, it can, in principle, be extended to diamagnetic proteins once a paramagnetic metal is attached to them by using specifically designed tags (43,44).…”

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confidence: 99%

Paramagnetic shifts in solid-state NMR of proteins to elicit structural information

Balayssac

Bertini

Bhaumik

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

141

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The recent observation of pseudocontact shifts (pcs) in 13 C highresolution solid-state NMR of paramagnetic proteins opens the way to their application as structural restraints. Here, by investigating a microcrystalline sample of cobalt(II)-substituted matrix metalloproteinase 12 [CoMMP-12 (159 AA, 17.5 kDa)], it is shown that a combined strategy of protein labeling and dilution of the paramagnetic species (i.e., 13 C-, 15 N-labeled CoMMP-12 diluted in unlabeled ZnMMP-12, and 13 C-, 15 N-labeled ZnMMP-12 diluted in unlabeled CoMMP-12) allows one to easily separate the pcs contributions originated from the protein internal metal (intramolecular pcs) from those due to the metals in neighboring proteins in the crystal lattice (intermolecular pcs) and that both can be used for structural purposes. It is demonstrated that intramolecular pcs are significant structural restraints helpful in increasing both precision and accuracy of the structure, which is a need in solid-state structural biology nowadays. Furthermore, intermolecular pcs provide unique information on positions and orientations of neighboring protein molecules in the solid phase.S olid-state NMR (SSNMR) on biomolecules is a rapidly growing technique, with an increased interest based on its ability to determine protein structures in the solid phase (1, 2) and to permit the study of noncrystalline biomolecular systems such as membrane proteins (3) and fibrils (4, 5).Limitations in biomolecular structural determination through SSNMR are due to the difficulties in obtaining a large number of restraints to be used for structural purposes (4, 6-9). Most of the structural information is obtained through distance restraints, analogous to nuclear Overhauser effects (NOE) in solution NMR, which in the solid state are obtained through experiments such as proton-driven spin diffusion (PDSD) (1, 6, 10), and CHHC (11), whereas specifically designed sequences can be applied on short peptides (4,(12)(13)(14). The ability to obtain a large number of distance restraints is hampered by the reduced resolution of SSNMR spectra, which increases the amount of ambiguities in the assigned restraints (15), whereas relayed transfer (10, 16) and the effects of the dipolar truncation (17) affect the accuracy of these restraints. These problems have been tackled by working on samples prepared with selective labeling schemes (1, 6) and, more recently, on uniformly labeled proteins with the help of software able to provide automated PDSD/ CHHC assignment and on dealing with a large number of ambiguous restraints (10,15,18). However, even when additional dihedral angle restraints from backbone chemical shiftsthrough Chemical Shift Index (CSI) (19) or TALOS (20) programs-are used, the size of the affordable proteins has been, up to now, limited to small systems (Ͻ100 aa) (10,15,18). In this work, we show how SSNMR paramagnetic restraints such as pseudocontact shifts (pcs) can be used as additional sources of restraints for protein structural determination, even providing information abou...

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“…The tags were synthesized in enantiomerically pure form to ensure that only a single stereoisomer is obtained following the ligation reaction with the target protein. 16,53 Ligation Reactions. The click reactions ( Figure 1) were performed at room temperature at pH 7.5 in 50 mM HEPES buffer, catalyzed by 0.2 mM Cu(I) ions in the presence of 5 mM sodium ascorbate and 1 mM of the Cu(I)-stabilizing ligand BTTAA.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

et al. 2013

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Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclenbased paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide−alkyne cycloaddition to a genetically encoded p-azido-L-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.

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

Cited by 134 publications

References 33 publications

Examination of Matrix Metalloproteinase-1 in Solution

Examination of Matrix Metalloproteinase-1 in Solution

Paramagnetic shifts in solid-state NMR of proteins to elicit structural information

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

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