Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: application to chromatin folding.
Quantitative analysis of chromatin electrophoretic mobility (mu) in agarose gels provides a measure of three structural parameters: average surface electrical charge density, which is proportional to the gel-free mu (mu 0), effective radius (Re), and particle deformability [Fletcher, T. M., Krishnan, U., Serwer, P., & Hansen, J. C. (1994) Biochemistry 33, 2226-2233]. To determine whether the intramolecular conformational changes associated with salt-dependent chromatin folding influence these electrophoretic parameters, defined oligonucleosomes were reconstituted from monodisperse tandemly repeated 5S DNA and varying amounts of histone octamers. These oligonucleosomes were subjected to both quantitative agarose gel electrophoresis and analytical velocity ultracentrifugation in buffers containing 0-2 mM MgCl2. Ionic conditions that caused a 40% increase in the oligonucleosome sedimentation coefficient (s20,w) also caused both a 30% decrease in Re and a 60% decrease in the magnitude of the mu 0. Furthermore, the Mg(2+)-dependent changes in s20,w, Re, and mu 0 each exhibited the same nonlinear dependence on the degree of nucleosome saturation of the DNA. These data demonstrate that quantitative agarose gel electrophoresis can be used to detect and characterize the process of chromatin folding. In addition, they suggest that this approach can be used for characterization of the conformational dynamics of many other types of macromolecular assemblies, including those systems that are not yet amenable for study by more traditional quantitative biophysical techniques.
Fletcher
T M
TM
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760.
Serwer
P
P
Hansen
J C
JC
D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D009707:Nucleosomes / Q000378:metabolism; D017510:Protein Folding
D002843:Chromatin; D046911:Macromolecular Substances; D009707:Nucleosomes; D015636:Magnesium Chloride; D004247:DNA