Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Nature. 2006 Apr 27;440(7088):1227-30. doi: 10.1038/nature04673.

Abstract

Macromolecular complex formation is governed by two opposing constraints of specificity and speed. Kinetic and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process or by the creation of a short-range attractive potential around the target site. This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Base Sequence
  • Biological Transport
  • DNA / chemistry
  • DNA / metabolism
  • Homeodomain Proteins / chemistry*
  • Homeodomain Proteins / metabolism*
  • Humans
  • Magnetic Resonance Spectroscopy / methods*
  • Magnetics*
  • Models, Molecular
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Neoplasm Proteins / chemistry*
  • Neoplasm Proteins / metabolism*
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Structure, Tertiary

Substances

  • HOXD9 protein, human
  • Homeodomain Proteins
  • Multiprotein Complexes
  • Neoplasm Proteins
  • DNA