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Jose K. James
B.S., Villanova University - 2009
Thesis Advisor: Vikas Nanda, Ph.D.
Graduate Program in Biomedical Engineering
CABM, Room 010
Friday, April 21, 2017
Tropomyosin (Tpm) is a protein involved in the regulation of the cell cytoskeleton. Minor sequence variations produce dramatic effects on Tpm folding resulting in clinical disorder and demonstrate a need to understand and model accurately how key residues globally impact Tpm structural stability and dynamics.
The structure of Tpm is a coiled-coil homodimer that displays upon denaturation multiple transitions corresponding to semi-independent cooperative folding domains. However, the exact boundaries that separate regional folding is unknown due to the continuous coiled-coil structure. By identifying local differences in folding and dynamics in Tpm the nature and architecture of these folding domains may be revealed.
From a simple helix-coil model, the intrinsic helicity of a given residue produces regions that allow for helical propagation or disfavor it. These latter insulating sequences can effectively decouple cooperativity between adjacent regions and create multiple folding domains. Hydrogen deuterium exchange measures with mass spectrometry (HDX-MS) local conformational fluctuations and identifies differences in helicity between three regions. The most dynamic middle area separates two more strongly helical terminal domains, concurrent with our model. A more detailed analysis of differences in conformation dynamics that underlie folding domains is done with molecular dynamics simulations. By reducing dynamics to a small collective coordinate space, a three domain dynamic structure is revealed in Tpm. The size and boundaries between low frequency dynamic domains roughly correspond with observations from HDX, suggesting that local conformational fluctuations detected by HDX are partly due to long range coordinated motions. Using MD simulations as a model of the inter-domain structure of Tpm, important deviations are discovered between mammalian and invertebrate species as well as disease causing mutants. Further study of the effects of these deviations from sequence may provide greater insight to Tpm structure and function.