Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes
Open Access
- 18 April 2013
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 8 (4), e57018
- https://doi.org/10.1371/journal.pone.0057018
Abstract
Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.This publication has 44 references indexed in Scilit:
- Dynamics of nucleosome remodelling by individual ACF complexesNature, 2009
- Probing Biomolecular Structures and Dynamics of Single Molecules Using In-gel Alternating-Laser ExcitationAnalytical Chemistry, 2009
- Nucleosome disassembly intermediates characterized by single-molecule FRETProceedings of the National Academy of Sciences of the United States of America, 2009
- spFRET Using Alternating Excitation and FCS Reveals Progressive DNA Unwrapping in NucleosomesBiophysical Journal, 2009
- Protein–Protein Förster Resonance Energy Transfer Analysis of Nucleosome Core Particles Containing H2A and H2A.ZJournal of Molecular Biology, 2007
- Fueling protein–DNA interactions inside porous nanocontainersProceedings of the National Academy of Sciences of the United States of America, 2007
- Single-Pair FRET Microscopy Reveals Mononucleosome DynamicsJournal of Fluorescence, 2007
- Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and ExperimentsThe Journal of Physical Chemistry B, 2006
- Rapid spontaneous accessibility of nucleosomal DNANature Structural & Molecular Biology, 2004
- New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioningJournal of Molecular Biology, 1998