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Methods for measuring rates of protein binding to insoluble scaffolds in living cells: Histone H1‐chromatin interactions
Author(s) -
Lele Tanmay,
Wagner Stefan R.,
Nickerson Jeffrey A.,
Ingber Donald E.
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20997
Subject(s) - chromatin , fluorescence recovery after photobleaching , histone , biophysics , kinetics , chemistry , reaction rate constant , dissociation constant , photobleaching , biochemistry , biology , fluorescence , dna , physics , receptor , quantum mechanics , membrane
Understanding of cell regulation is limited by our inability to measure molecular binding rates for proteins within the structural context of living cells, and many systems biology models are hindered because they use values obtained with molecules binding in solution. Here, we present a kinetic analysis of GFP‐histone H1 binding to chromatin within nuclei of living cells that allows both the binding rate constant k ON and dissociation rate constant k OFF to be determined based on data obtained from fluorescence recovery after photobleaching (FRAP) analysis. This is accomplished by measuring the ratio of bound to free concentration of protein at steady state, and identifying the rate‐determining step during FRAP recovery experimentally, combined with mathematical modeling. We report k OFF = 0.0131/s and k ON = 0.14/s for histone H1.1 binding to chromatin. This work brings clarity to the interpretation of FRAP experiments and provides a way to determine binding kinetics for nuclear proteins and other cellular molecules that interact with insoluble scaffolds within living cells. J. Cell. Biochem. 99: 1334–1342, 2006. © 2006 Wiley‐Liss, Inc.