Calorimetric Determination of Dystrophin ABD1 Unfolding Energetics
2018-12
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Calorimetric Determination of Dystrophin ABD1 Unfolding Energetics
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2018-12
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Muscular Dystrophy (MD) is a disease that effects the structural integrity of muscle cells. Studies have linked the Dystrophin protein to MD as the most commonly altered gene in patients with MD. Using empirical models established to predict the change in heat capacity associated with protein unfolding, we we correlated the likelihood of a mutation as being disease causing with an associated change in the heat capacity at that amino acid position. These studies focused on the first Actin Binding Domain of Dystrophin (ABD1, 27kDa) using Differential Scanning Calorimetry (DSC), as it is the region with the highest density of disease causing mutations. ABD1 is comprised of two Calmodulin Homology domains (CH1 and CH2) connected by a short linker region and is predicted to be slightly disordered. [1] Analyzing the data acquired from DSC proved to be rather difficult as it was highly dependent on the baseline definition, which can be rather noisy. This thesis describes the evolution of our DSC analysis starting with an analysis published in the Biophysical Journal. This method suggested the a change in heat capacity (∆Cp of 5 ± 5 kcal/mol). However, the model showed some systematic deviation from the experimental data so the data was fit to a Gaussian and Hubbert distribution. Then a deconvolution approach revealed the presence of an appreciable occupancy (approximately 50%) of intermediate states that helps account for the deviations from a two-state model. Deconvoluting the transitions revealed at least one intermediate transition with ∆G(37◦C) of 2 ± 1kcal/mol and an unfolding free energy of 2.2 ± 0.6kcal/mol and a change in heat capacity that is smaller than predicted. This free energy is comparable to that which has been determined for actin binding thus implicating unfolding of ABD1 upon binding actin, possibly through separating the CH domains or some other mechanism.
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University of Minnesota M.S. thesis. December 2018. Major: Chemistry. Advisor: Anne Hinderliter. 1 computer file (PDF); xvi, 81 pages.
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Coffman, Christian. (2018). Calorimetric Determination of Dystrophin ABD1 Unfolding Energetics. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/202099.
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