Exploring the allowed space for solution NMR studies of membrane proteins.

摘要

This thesis explores the general applicability of solution NMR methods to solve specific questions in membrane structural biology.; Based on the completed total genomic sequence analyses of microorganisms, membrane proteins account for about one-third of all proteins. However, despite the recent explosive advances in structural biology the number of membrane protein structures solved each year remains minimal. Diacylglycerol kinase (DAGK), a 13 kD integral membrane protein with three trans-membrane helices, was examined as a model membrane protein in a wide variety of micelles, organic solvent mixtures, and amphipathic polymers. In each system we examined DAGK's oligomeric state, motional properties, and the degree to which DAGK resembles the functional bilayer-associated structure. Our results indicated that while organic solvent systems yielded the best NMR spectra, they were disruptive of DAGK's structure. In the case of aqueous detergent micelles the results indicated that medium-sized-chain detergents are better than short-chain detergents for maintaining native-like protein conformations. We also observed that both short and long-chained detergents led to detergent-protein aggregates of similar size, placing detergent solubilized DAGK well beyond the molecular weight limit for NMR based structural determination. Conditions were found in which amphipol-solubilized DAGK maintained detectable catalytic activity while also exhibiting a translational diffusion coefficient suggestive of a lower effective molecular weight for the enzyme than in any micellar system. These observations provide the groundwork for further development of amphipol systems.; In the second part of this thesis the ability of the membrane-mimicking system to induce ordered structure in the cytoplasmic juxta-membrane domain of the Adenovirus E3-13.7 protein was investigated. This protein perturbs normal sorting of certain host receptors. The E3 cytoplasmic domain was observed to exist in a mostly random structural state in aqueous solution, but associated with high affinity to dodecylphosphocholine micelles, where it adopted an ordered structure. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. Binding was independent of the presence of an N-terminal myristoyl chain.