The Interaction between Rac1 and Its Guanine Nucleotide Dissociation Inhibitor (GDI), Monitored by a Single Fluorescent Coumarin Attached to GDI
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- 作者：Anthony R. Newcombe ; Richard W. Stockley ; Jackie L. Hunter ; and Martin R. Webb
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 25, 1999
- 年：1999
- 卷：38
- 期：21
- 页码：6879 - 6886
- 全文大小：78K
- 年卷期：v.38,no.21(May 25, 1999)
- ISSN：1520-4995
文摘
The interaction of rac with guanine nucleotide dissociation inhibitor protein (rhoGDI) isdescribed, using GDI fluorescently labeled on its single cysteine with N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC). The labeled GDI shows a 70% decrease in fluorescenceemission on binding geranylgeranylated rac1·GDP and has an affinity for rac1 within a factor of 2 of theunlabeled GDI. The labeled GDI was used to determine the kinetic mechanism of the interaction bymeasuring the association and dissociation in real time. The kinetics are interpreted in terms of a two-stepmechanism: binding of rac to GDI and then a conformational change of the complex with an overalldissociation constant of 0.4 nM. The conformational change has a rate constant of 7.3 s-1 (pH 7.5, 30
C), and the reverse has a rate constant of 1.4 × 10-3 s-1. To overcome difficulties inherent in using andmanipulating lipid-modified rac, we also used a combination of unmodified rac1, expressed in Escherichiacoli and produced with C-terminal truncation (thus lacking the cysteine that is the site of lipid attachment),and farnesylated C-terminal peptide. This combination can mimic geranylgeranylated rac1, producing acomplex with the coumarin-labeled GDI, and was used to examine the relative importance of differentregions of rac1 in interaction with GDI.
C), and the reverse has a rate constant of 1.4 × 10-3 s-1. To overcome difficulties inherent in using andmanipulating lipid-modified rac, we also used a combination of unmodified rac1, expressed in Escherichiacoli and produced with C-terminal truncation (thus lacking the cysteine that is the site of lipid attachment),and farnesylated C-terminal peptide. This combination can mimic geranylgeranylated rac1, producing acomplex with the coumarin-labeled GDI, and was used to examine the relative importance of differentregions of rac1 in interaction with GDI.