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Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins
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Detecting transient intermediates in macromolecular binding by paramagnetic NMR.

Junji Iwahara1, G Marius Clore

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.

Nature
|April 28, 2006
PubMed
Summary
This summary is machine-generated.

Researchers used intermolecular paramagnetic relaxation enhancement (PRE) to detect transient intermediates in macromolecular binding. This method reveals how transcription factors find specific DNA sites faster by interacting with non-cognate sites.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Macromolecular complex formation balances specificity and speed.
  • Rate enhancement in binding kinetics can involve reduced search dimensionality or attractive potentials.
  • Transient, non-specific binding intermediates are implied by kinetic and theoretical models.

Purpose of the Study:

  • To directly detect and characterize low-population transient intermediates in macromolecular binding under equilibrium.
  • To investigate the role of non-specific DNA binding in enhancing the association rate of sequence-specific transcription factors.
  • To demonstrate the utility of intermolecular paramagnetic relaxation enhancement (PRE) for studying binding dynamics.

Main Methods:

  • Utilized intermolecular paramagnetic relaxation enhancement (PRE) to probe transient interactions.
  • Applied PRE to study the binding of the HOXD9 homeodomain transcription factor to DNA.
  • Analyzed PRE data in the fast exchange regime to characterize intermediate structures and dynamics.

Main Results:

  • Directly detected transient, low-population intermediates during transcription factor-DNA binding.
  • Observed that these intermediates, formed at non-cognate DNA sites, share structural similarity with the specific complex.
  • Characterized two distinct search mechanisms involving intra- and intermolecular translocations.
  • Demonstrated that non-specific binding enhances the rate of specific DNA association.

Conclusions:

  • Intermolecular PRE is a powerful technique for detecting and characterizing transient binding intermediates.
  • Transcription factors utilize non-specific DNA interactions as a search strategy to accelerate specific binding.
  • The methodology is broadly applicable to diverse macromolecular binding processes.