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Conformational Transitions of an Intrinsically-Disordered Protein: Effects of Environment and Binding Partners on Myelin Basic Protein

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Title: Conformational Transitions of an Intrinsically-Disordered Protein: Effects of Environment and Binding Partners on Myelin Basic Protein
Author: De Avila, Miguel
Department: Department of Molecular and Cellular Biology
Program: Biophysics
Advisor: Harauz, George
Abstract: Central nervous system myelin is composed of many essential proteins that facilitate the structure, function, and compaction of myelin, and among these members are the developmentally-regulated myelin basic proteins (MBPs), which arise from the Golli (Gene of Oligodendrocyte Lineage) complex. Although MBPs play a key role in myelin compaction, they are also intrinsically-disordered, multifunctional proteins with a variety of post-translational modifications (PTMs). These modifications can further facilitate a variety of biological interactions including binding to proteins including actin, tubulin, calmodulin, and SH3- domain proteins. Classic MBP isoforms contain a proline- rich region comprising amino acids 92-99 (murine sequence -TPRTPPPS-) which contains a minimal SH3-ligand domain. This thesis is an investigation of the structural elements of 18.5-kDa MBP in the presence of membrane-mimetic environments, such as trifluoroethanol (TFE) or dodecylphosphocholine (DPC) micelles, and in the presence of its binding partners calmodulin and Fyn tyrosine kinase. A divide-and-conquer approach was used to study the structure and interactions of 18.5-kDa MBP by solution NMR spectroscopy. The structural propensity of the central region of MBP (S72-S107) was investigated in the presence of DPC, and the propensity of the proline-rich region to adopt a poly-proline type II (PPII) conformation is correlated to the α-helical stability of the central region. Investigations of the mode of interaction of MBP peptides with the SH3-domain of Fyn determined that the proline-rich region of MBP relies on cooperative binding by secondary binding site 30 residues away. Investigations of the mode of interaction with calmodulin showed the interaction is calcium-dependent, and influences the structural propensity of the entire peptide. The investigations of structural propensities were extended beyond peptide-based experiments to collect structural restraints on the global fold of MBP in the presence of 30% TFE. Spin-labeled MBP was used to determine paramagnetic relaxation enhancements of previously assigned nuclei. The experiments showed contamination by diamagnetic signals in the oxidized sample, and intermolecular relaxation. Despite this, the trend of intensity ratios observed was in good agreement with current ongoing Förster resonance energy transfer (FRET) experiments, which will be used as additional restraints to triangulate the structural model of full-length MBP in membrane-mimetic conditions.
URI: http://hdl.handle.net/10214/8322
Date: 2014-08
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada