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生物化学
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Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins
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生物化学
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JoVE 杂志 生物化学
Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

DOI:
10.3791/63057-v

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07:24 min

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September 23, 2021

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,
1Department of Biochemistry and Structural Biology and Greehey Children’s Cancer Research Institute,The University of Texas Health Science Center at San Antonio

Chapters

  • 00:05Introduction
  • 00:49Conjugating the 3-Maleimido-PROXYL Nitroxide Spin Label
  • 01:38Preparing the NMR Sample for Measuring Intramolecular PRE
  • 02:11Setting Up the NMR Spectrometer and Experiment Specific Parameters
  • 03:51Setting Up the 1HN–T2 Experiment
  • 04:58Making a Diamagnetic Sample by Reducing Spin-Label with Ascorbic Acid
  • 05:52Results: 1HN–T2 Rates for Residues 171 – 264 of the Intrinsically Disordered Domain of EWSR1
  • 06:30Conclusion

Summary

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A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Tags

Keywords: Paramagnetic Relaxation EnhancementIntrinsically Disordered ProteinsBiomolecular InteractionsNMR TechniquesSpin Label15N Isotopically Labeled ProteinGel FiltrationEllman’s ReagentNMR Sample PreparationPulse Calibration

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