Graduate Thesis Or Dissertation
 

Conformational transitions of nucleosome core particles monitored with time-resolved fluorescence spectroscopy

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  • Time-resolved fluorescence spectroscopy was used to monitor the effects of varying ionic strength on nucleosome core particle structure. Two main methods were used in these studies. First, the fluorescence anisotropy decay of bound ethidium was measured and was shown to reflect the rotational tumbling of the core particle through solution, the longest recovered decay time being a measure of the rotational correlation time of the particle. A rotational correlation time of 165 ns was recovered for the native core particle at 10 mM ionic strength, in excellent agreement with that predicted by hydrodynamic calculations based on the particle's known size and shape. This technique was then used to measure the rotational correlation time of the core particle as a function of ionic strength. Below 1 mM salt the recovered rotational correlation times suggested little change in shape throughout the region of the reversible low salt transition. At very low ionic strengths (below 0.2 mM), where the low salt transition becomes irreversible, the rotational correlation time increased sharply to ~330 ns, suggesting a major change in the core particle structure. Computer modeling was performed to show that this increase was most likely due to a substantial elongation in the core particle structure, to at least a 5:1 axial ratio. At elevated ionic strengths, the rotational correlation time was seen to increase from the initial value of ~165 ns to ~240 ns as the salt concentration was raised from 10 mM to 0.35 M, with further increases being observed only above 0.65 M; we term this initial increase the moderate salt transition. Trypsinization of the core particles to remove the Nterminal histone domains completely abolished the increase, demonstrating that the moderate salt transition as measured by this technique involves the release of these protein domains from the body of the core particle. The second method used involved the measurement of the fluorescence decay of the intrinsic tyrosine residues of the core particle. This decay proved to be very complex, and was best represented by a distribution of lifetimes, suggesting different environments for the tyrosines. This distribution changed as the ionic strength of the solution changed, suggesting the movement of tyrosine residues to differing environments as the particle undergoes the low and moderate salt transitions, as well as the high salt dissociation.
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