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|Title: ||Thermal Fluctuation Spectroscopy And Its Application In The Study Of Biomolecules|
|Authors: ||Nagapriya, K S|
|Advisors: ||Raychaudhuri, A K|
|Keywords: ||Energy Fluctuations|
Thermal Fluctuation Spectroscopy (TFS)
Biomolecules - Denaturation
DNA - Thermal Fluctuations
Histones - Thermal Fluctuations
Nucleosomes - Thermal Fluctuations
|Submitted Date: ||Aug-2006|
|Series/Report no.: ||G20909|
|Abstract: ||The aim of this thesis is to study the energy fluctuations (leading to thermal fluctuations) during thermal and enzymatic denaturation of biological molecules and to study the variation in fluctuations between simple molecules like the DNA (which have only a secondary structure) to molecules with higher order structures and packaging. We have developed a new technique - Thermal Fluctuation Spectroscopy (TFS) to study these fluctuations.
The technique of Thermal Fluctuation Spectroscopy (TFS) is a combination of microcalorimetry and noise measurement techniques. The combination of these two powerful techniques has never been exploited before. In this technique any energy exchange between sample and the substrate is reflected as a thermal fluctuation of the substrate. The system resolution is few parts per billion (ppb) and fluctuations in energy ~ 100nJ (which correspond to temperature fluctuations ~ K) can be measured.
Chromatin is the basic building block of chromosome and this thesis focuses on the constituents this fundamental building block - DNA, histones and nucleosomes.
Heteropolymeric dsDNA shows extremely large non-Gaussian fluctuation around its melting temperature. For homopolymeric DNA the fluctuations during denaturation are smaller. The thermal fluctuation during denaturation of a heteropolymer in buffer is several orders larger than when the DNA is on a substrate while that for a homopolymer is comparable in both cases. Our measurements established that heteropolymeric dsDNA denaturation occurs in two stages. Initially, at around 330 K, bubbles are formed in the AT rich regions. At higher temperatures, the GC rich regions binding them denature in a cooperative transition causing extremely large fluctuations.
TFS on histone monomers showed that H1 monomer shows an increase in thermal fluctuation in the temperature range studied, while the core histones did not. We infer that this is due to the fact that the core histones may not be properly folded when they exist as monomers. It was seen that H1 crosses an energy barrier of 17 kcal/mol to go from its native to denatured state. The transition was kinetically driven with a fixed barrier till 352 K. At 352K, the barrier softened by ~ 1 kcal/mol leading to faster denaturation. The core histones when assembled as dimers/oligomers showed an increase in fluctuation at temperatures below 350 K.
The assembling of these histones and DNA into a mononucleosome causes a very large increase in fluctuation over the entire temperature range studied. TFS showed that the fluctuation during mononucleosome denaturation was much larger than a simple sum of the fluctuations of its constituents. From the data we were able to identify that the denaturation starts with dissociation and unfolding of the core histones and the denaturation of AT rich regions of the DNA which leads to the breaking of some of the histone-DNA contacts. At higher temperatures the linker histone H1 and the GC rich regions of the DNA denature, leading to a collapse of the entire nucleosome structure. The broadness of the transition region (the fact that the fluctuation is large over the entire temperature range) was attributed to the presence of different types of contacts and interactions (with different energies) stabilizing the nucleosome structure. The nucleosome was found to favour large energy jumps over smaller ones indicating that the denaturation has an element of cooperativity involved.
Using TFS we have been able to determine the fluctuations involved in the denaturation of biomolecules like DNA, histones and nucleosomes. The energy barriers to denaturation have been determined. We have also been able to give models for the denaturation of these biomolecules. We have also shown that it is possible to study enzymatic digestion using TFS. Thus, the technique of TFS is a viable tool for the study of fluctuations in reactions, in biomolecules, during transitions and in any process where there is an energy exchange involved.|
|Appears in Collections:||Physics (physics)|
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