title: Methods for measuring rates of protein binding to insoluble scaffolds in living cells: histone H1-chromatin interactions.
abstract: 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.
authors.affiliation: Department of Pathology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Tanmay
authors.initials: T
authors.lastname: Lele
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Wagner
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Nickerson
authors.forename: Donald E
authors.initials: DE
authors.lastname: Ingber
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008433:Mathematics; D051379:Mice; D008962:Models, Theoretical; D041681:NIH 3T3 Cells; D011485:Protein Binding / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D006657:Histones; D011993:Recombinant Fusion Proteins