Methods for measuring rates of protein binding to insoluble scaffolds in living cells: Histone H1‐chromatin interactions

Tanmay Lele; Stefan R. Wagner; Jeffrey A. Nickerson; Donald E. Ingber

doi:10.1002/jcb.20997

journal article Jun 22, 2006

Methods for measuring rates of protein binding to insoluble scaffolds in living cells: Histone H1‐chromatin interactions

Tanmay Lele Stefan R. Wagner Jeffrey A. Nickerson Donald E. Ingber

Journal of Cellular Biochemistry Vol. 99 No. 5 pp. 1334-1342 · Wiley

View at Publisher Save 10.1002/jcb.20997

Abstract

AbstractUnderstanding 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 kON and dissociation rate constant kOFF 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 kOFF = 0.0131/s and kON = 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.

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Details

Published: Jun 22, 2006
Vol/Issue: 99(5)
Pages: 1334-1342
License: View

Authors

Department of Electrical and Computer Engineering, Aarhus University, 8200 Aarhus, Denmark

Cite This Article

Tanmay Lele, Stefan R. Wagner, Jeffrey A. Nickerson, et al. (2006). Methods for measuring rates of protein binding to insoluble scaffolds in living cells: Histone H1‐chromatin interactions. Journal of Cellular Biochemistry, 99(5), 1334-1342. https://doi.org/10.1002/jcb.20997

Methods for measuring rates of protein binding to insoluble scaffolds in living cells: Histone H1‐chromatin interactions

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