The proposed research centers on two aspects of polypeptide folding and assembly that are highly significant from both fundamental and biomedical perspectives. One focus involves amyloid, an aggregated protein state that is associated with many human diseases. The other focus is quaternary interactions involving single-pass transmembrane helices, which are important for signal transduction via cell-surface receptors. Atomic-resolution characterization of amyloids is extremely challenging because polypeptides in amyloids are insufficiently ordered for high-resolution diffraction (amyloids are not crystals), but the solid nature of amyloids precludes the use of powerful solution-state NMR and other biophysical methods. Remarkable progress has recently been made via solid-state NMR in terms of atomic-resolution models for specific amyloids, revealing an essentially infinite quaternary structure that differs from the discrete structural motifs within folded proteins. The first high-resolution glimpses of amyloid structure have raised questions that seem not to be fully answerable via the study of amyloids themselves. We are therefore pursuing a new approach based on soluble models for the amyloid state. The connection between natural amyloids and associated diseases is not clear: toxicity could arise from fibrils themselves, from oligomeric precursors, or both (most current literature favors soluble oligomers as the main toxic agents). Whatever the origin(s) of pathological effects, it is vital to acquire a fundamental understanding of the factors that influence structure and stability in the amyloid state. Since amyloids themselves are not amenable to many of the powerful strategies available for physical characterization of soluble proteins, we seek soluble model systems that manifest key features associated with authentic amyloid structures. Associations between single-pass transmembrane ?-helices (SPTM helices) are crucial for the function of many membrane proteins. Bitopic receptors, for example, contain single-pass helices that link the sensory extracellular domain with a functional intracellular domain. Signaling generally requires discrete receptor assemblies (dimers, trimers or larger oligomers). Formation of signaling-competent assemblies depends at least in part on specific interactions among SPTM helices. NMR-based structural models for SPTM helix assemblies have appeared in recent years, but there are no crystallographic data for bitopic receptor-derived SPTM helix assemblies, to our knowledge. The only relevant crystal structures involve the SPTM segment of the influenza M2 proton channel, which forms a tetramer, and very recent structures of the TM helix from DAP12, an immunoreceptor adaptor protein. Our long-term goal is to acquire multiple crystal structures for diverse SPTM helix assemblies and thereby contribute to a communal elucidation of the rules that govern intramembrane helix-helix recognition. Racemate and quasiracemate crystallization are major tools in our effort. Crystal structures of SPTM helix assemblies from bitopic receptors would be extremely significant complements to NMR-based models. The nature of the inter-helical interfaces is the most burning question, and crystal structures would provide valuable insights that may not be available via NMR.
The proposed research focuses on two very difficult and biomedically significant problems in protein structure. One effort involves aggregated forms of proteins known as 'amyloids', which are associated with many human diseases (e.g., Alzheimer's Disease, Parkinson's Disease). The other effort involves small segments of cell-surface proteins that are embedded in membranes and play crucial roles in information transfer into the cell. Mutations in these segments are linked to many human disease. For both topics, the link between structure and disease is unclear, and our studies are intended to contribute fundamental insights that might ultimately provide a foundation for therapeutically-oriented efforts.
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