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

Authors

Xun Cheng Su, Australian National University
Slobodan Jergic, University of WollongongFollow
Kiyoshi Ozawa, University of WollongongFollow
Nicolas Dale Burns, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

16853

Publication Details

Su, X. Cheng., Jergic, S., Ozawa, K., Burns, N. D., Dixon, N. E. & Otting, G. (2007). Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation. Journal of Biomolecular N M R, 38 (1), 65-72.

Abstract

The use of ¹⁵N-relaxation data for determination of the dissociation constant of a protein–protein complex is proposed for the situation where a ¹⁵N-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 ¹⁵N-labeled fragment of the C-terminal domain of the τ subunit (15 kDa) and the unlabeled α subunit (130 kDa).