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

Xun Cheng Su; Slobodan Jergic; Kiyoshi Ozawa; Nicolas Dale Burns; Nicholas E. Dixon; Gottfried Otting

doi:10.1007/s10858-007-9147-9

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

Xun Cheng Su, Slobodan Jergic, Kiyoshi Ozawa, Nicolas Dale Burns, Nicholas E. Dixon, Gottfried Otting^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

19 Citations (Scopus)

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).

Original language	English
Pages (from-to)	65-72
Number of pages	8
Journal	Journal of Biomolecular NMR
Volume	38
Issue number	1
DOIs	https://doi.org/10.1007/s10858-007-9147-9
Publication status	Published - May 2007

Access to Document

10.1007/s10858-007-9147-9

Cite this

@article{c44627dc96fe42ca92c7a375c3ccc638,

title = "Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation",

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).",

keywords = "Chemical exchange, Dissociation constant, E. coli DNA polymerase III, N relaxation, Protein-protein complex, Subunits α and τ",

author = "Su, {Xun Cheng} and Slobodan Jergic and Kiyoshi Ozawa and Burns, {Nicolas Dale} and Dixon, {Nicholas E.} and Gottfried Otting",

year = "2007",

month = may,

doi = "10.1007/s10858-007-9147-9",

language = "English",

volume = "38",

pages = "65--72",

journal = "Journal of Biomolecular NMR",

issn = "0925-2738",

publisher = "Springer Netherlands",

number = "1",

}

TY - JOUR

T1 - Measurement of dissociation constants of high-molecular weight protein-protein complexes by transferred 15N-relaxation

AU - Su, Xun Cheng

AU - Jergic, Slobodan

AU - Ozawa, Kiyoshi

AU - Burns, Nicolas Dale

AU - Dixon, Nicholas E.

AU - Otting, Gottfried

PY - 2007/5

Y1 - 2007/5

N2 - 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).

AB - 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).

KW - Chemical exchange

KW - Dissociation constant

KW - E. coli DNA polymerase III

KW - N relaxation

KW - Protein-protein complex

KW - Subunits α and τ

UR - http://www.scopus.com/inward/record.url?scp=34248547901&partnerID=8YFLogxK

U2 - 10.1007/s10858-007-9147-9

DO - 10.1007/s10858-007-9147-9

M3 - Article

SN - 0925-2738

VL - 38

SP - 65

EP - 72

JO - Journal of Biomolecular NMR

JF - Journal of Biomolecular NMR

IS - 1

ER -

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

Abstract

Access to Document

Other files and links

Fingerprint

Cite this