The Structure of Micelle-bound Proamylin and Advances in Nuclear Overhauser Effect Spectroscopy

Solution-state NMR spectroscopy provides an invaluable tool to study both the structure and dynamics of biomolecules in their near-native environment. This method has particular strength when studying intrinsically disordered proteins or systems with conformational heterogeneity. In contrast, solution NMR suffers with large molecular weight proteins due to spectral overlap and line broadening. This collection of work reports on the structure of an intrinsically disordered peptide prohormone bound to a membrane mimetic and challenges the molecular size problem with the development of a new experimental method to improve NOESY spectral resolution. In the first part, I describe the use of NMR and other biophysical methods in the thorough structural characterization of pro-islet amyloid polypeptide in a membrane environment. We mapped the protein-lipid complex interface, and examine the effects of membrane curvature, charge, and pH on complex formation. Surprisingly, we found that the C-terminal pro-hormone segment of this protein can fold and associate to membranes independently of the rest of the molecule, and comment on potential implications. The second part describes the development of the super-resolution NOESY experiment to improve data quality for protein structure determination. We use two model systems, ubiquitin and the hemagglunitin fusion peptide of the influenza A virus (HAfp) bound to large bicelles, in order to characterize the optimal experimental parameters with respect to molecular size for maximum resolution enhancement. Furthermore, we explain the theory, pitfalls, and contrast to apodization techniques.