| Structural studies of membrane proteins are essential to improve our understanding of cell-surface phenomena, however, they present a major challenge, since membrane proteins need to be studied in their native environment, a lipid bilayer. Field oriented phospholipid bilayer disks, or bicelles, can be used to mimic biological membranes, while allowing unique NMR measurements of structural and dynamic properties for molecules that are partially oriented through their association with the bilayer. This method has been successfully used in the study of membrane-bound carbohydrates. The work presented here represents the efforts undertaken to extend this methodology to the study of membrane associated peptides.;A protein that associates with membranes in a GTP dependent manner was selected as the system of study. ARF1, an N-myristoylated protein, plays an important role in membrane trafficking. Its interaction with membranes, although highly studied, remains not well understood. Site specifically 13C and 15N labeled peptides were synthesized, with and without a lipid anchor, based on the sequence of the N-terminal amphipathic helix of the ARF1 protein. These peptides were successfully incorporated into micelle and bicelle lipid systems, and their secondary structure was studied in the isotropic micelle solutions by CD and solution NMR spectroscopy.;The degree of order for bicelle associated molecules is too high to allow conventional proton detected NMR experiments, but through the use of heteronuclear observe solid-state NMR techniques it is possible to measure anisotropic NMR spectral parameters. We extended the previously used NMR methods by optimizing 15N observe experiments, and by using 2-dimensional separated local field spectroscopy. Dipolar couplings and CSA data from both the backbone and aromatic side chains were measured by carbon-13 and nitrogen-15 observe 1D and 2D NMR experiments. Order-matrix calculations were used to analyze the anisotropic spin interaction data. Numerical methods that make this analysis fast and more convenient were developed and are presented.;In summary, through the use of solid-sate NMR techniques, the behavior of membrane associated peptides on the surface of a bicelle has been characterized. Effects of the lipid anchor and the behavior of the aromatic side chains are discussed in the context of the ARF1 biochemistry. |
