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Related Experiment Videos

Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.

Michael D Purdy1, Pinghua Ge, Jiyan Chen

  • 1Interdisciplinary Program in Biophysics, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville 22908, USA.

Acta Crystallographica. Section D, Biological Crystallography
|June 22, 2002
PubMed
Summary

Lanthanide chelates like DTPA-PDPH can be incorporated into protein crystals, providing a strong anomalous signal for X-ray crystallography phase determination. This method aids in solving complex protein structures, though its broad applicability for high-throughput studies is limited.

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Area of Science:

  • Biophysical Chemistry
  • Structural Biology
  • Crystallography

Background:

  • Lanthanides offer a significant anomalous signal for X-ray crystallography, crucial for phase determination in biological macromolecules.
  • Incorporating lanthanides into protein crystals requires specific chemical strategies.

Purpose of the Study:

  • To synthesize and evaluate thiol-reactive lanthanide chelates for labeling proteins.
  • To assess the utility of these chelates in X-ray crystallography, particularly for phase determination.

Main Methods:

  • Synthesis of diethylenetriaminepentaacetic 3-(2-pyridyldithio)propionyl hydrazide (DTPA-PDPH) and 1,4,7,10-tetraazacyclododecane-N,N",N",N"-tetraacetic 3-(2-pyridyldithio)propionyl hydrazide (DOTA-PDPH).
  • Labeling of a cysteine mutant of phosphate-binding protein (PBP-A197C) with DTPA-PDPH loaded with dysprosium (Dy).

Related Experiment Videos

  • X-ray diffraction data collection and Single-wavelength Anomalous Diffraction (SAD) phasing using synchrotron radiation.

    • Main Results:

    • Successful labeling of PBP-A197C with DTPA-PDPH-Dy, confirmed by mass spectrometry and SDS-PAGE.
    • Crystallization of labeled protein was identical to unlabeled protein, yielding a strong anomalous signal.
    • SAD phasing using Dy L(III) edge data provided high-quality electron-density maps, enabling automated model building.
    • The method's generality was tested on other proteins, with variable success due to solubility and crystallization issues.

    Conclusions:

    • Thiol-reactive lanthanide chelates are effective for introducing a strong anomalous signal into protein crystals for SAD phasing.
    • While not ideal for high-throughput crystallography, this method is valuable for challenging protein structure determination cases.
    • Further optimization may be needed to overcome limitations in protein solubility and crystallization observed in some instances.