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Related Experiment Videos

A caged lanthanide complex as a paramagnetic shift agent for protein NMR.

Miguel Prudêncio1, Jan Rohovec, Joop A Peters

  • 1Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Chemistry (Weinheim an Der Bergstrasse, Germany)
|June 30, 2004
PubMed
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A new lanthanide complex, caged lanthanide NMR probe (CLaNP), enables detailed protein characterization using paramagnetic NMR spectroscopy. This probe facilitates long-range distance measurements for enhanced structural analysis.

Area of Science:

  • Biochemistry
  • Chemical Biology
  • Structural Biology

Background:

  • Paramagnetic NMR spectroscopy is a powerful tool for protein characterization.
  • Existing probes often suffer from high metal mobility, limiting structural information.
  • There is a need for robust probes that provide accurate long-range distance restraints.

Purpose of the Study:

  • To develop and characterize a novel lanthanide complex, CLaNP, for protein structure determination.
  • To assess the utility of CLaNP in paramagnetic NMR spectroscopy for measuring pseudocontact shifts and residual dipolar couplings.
  • To evaluate the impact of CLaNP attachment on protein structure and dynamics.

Main Methods:

  • Synthesis of a lanthanide complex (CLaNP) chelated by a DTPA derivative with thiol-reactive groups.

Related Experiment Videos

  • Bidentate attachment of CLaNP to engineered Cys residues on proteins.
  • Paramagnetic NMR spectroscopy experiments to measure pseudocontact shifts and residual dipolar couplings.
  • X-ray crystallography to determine the structure of the probe-protein complex.
  • Comparison with diamagnetic control experiments.

    • Main Results:

    • CLaNP was successfully synthesized and attached to proteins via engineered Cys residues.
    • Bidentate attachment significantly reduced metal-protein dynamics, enabling accurate pseudocontact shift measurements.
    • The probe induced measurable pseudocontact shifts up to 40 Å from the metal.
    • High magnetic field alignment of the probe-protein complex resulted in residual dipolar couplings.
    • Protein structure remained unaffected by CLaNP attachment, as confirmed by crystallography and control experiments.
    • Multiple isomeric forms of CLaNP provided diverse paramagnetic tensors for enhanced structural restraints.

    Conclusions:

    • CLaNP is an effective probe for protein characterization by paramagnetic NMR spectroscopy.
    • The probe's design facilitates accurate long-range distance measurements, aiding structural elucidation.
    • CLaNP offers a versatile tool for obtaining structural information without perturbing native protein structures.