Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation

J Biomol NMR. 2007 May;38(1):65-72. doi: 10.1007/s10858-007-9147-9. Epub 2007 Mar 28.

Abstract

The use of (15)N-relaxation data for determination of the dissociation constant of a protein-protein complex is proposed for the situation where a (15)N-labeled protein is bound to an unlabeled protein of high molecular weight, and the chemical exchange between bound and free protein is fast on the NMR time scale. The approach is shown to be suitable for estimating dissociation constants in the micromolar to millimolar range, using protein solutions at relatively low concentration. An example is shown for the interaction between two subunits from the Escherichia coli DNA polymerase III complex, involving a (15)N-labeled fragment of the C-terminal domain of the tau subunit (15 kDa) and the unlabeled alpha subunit (130 kDa).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Polymerase III / chemistry
  • DNA Polymerase III / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Kinetics
  • Molecular Weight
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Binding
  • Proteins / chemistry*
  • Proteins / metabolism*

Substances

  • Escherichia coli Proteins
  • Nitrogen Isotopes
  • Proteins
  • DNA Polymerase III