文章基本信息

标题：Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells
本地全文：下载
作者：Bin Wu ; Yan Chen ; Joachim D. Müller 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：9
页码：4117-4122
DOI：10.1073/pnas.0905670107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Measuring the binding curve and stoichiometry of protein complexes in living cells is a prerequisite for quantitative modeling of cellular processes. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions, but lacks suitable methods for quantifying protein heterointeractions in the cell. We address this challenge by introducing heterospecies partition (HSP) analysis for protein heterointeractions of the type D + nA{leftrightarrow}DAn. HSP directly identifies the heterointeracting species from the sample mixture and determines the binding curve and stoichiometry of the protein complex. The HSP method is applied to provide the first direct characterization of the ligand-dependent binding of the retinoic X receptor to the coactivator transcription intermediate factor 2. A previous study based on protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. This study provides proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein heterointeractions by dual-color fluorescence fluctuation spectroscopy in living cells.
关键词：coactivator ; fluorescence correlation spectroscopy ; fluorescence fluctuation spectroscopy ; nuclear receptor ; time-integrated fluorescence cumulant analysis