Structure and stability in proteins and analogues

 描述（由申请人提供）：拟议的研究集中在多肽折叠和组装的两个方面，从基础和生物医学的角度来看，这两个方面都非常重要。一个焦点涉及淀粉样蛋白，一种与许多人类疾病相关的聚集蛋白状态。另一个焦点是涉及单程跨膜螺旋的四级相互作用，这对通过细胞表面受体的信号转导很重要。淀粉样蛋白的原子分辨率表征极具挑战性，因为淀粉样蛋白中的多肽对于高分辨率衍射来说不够有序（淀粉样蛋白不是晶体），但淀粉样蛋白的固体性质排除了使用强大的溶液状态NMR和其他生物物理方法。最近通过固态NMR在特定淀粉样蛋白的原子分辨率模型方面取得了显着的进展，揭示了一个基本上无限的四级结构，不同于折叠蛋白质内的离散结构基序。淀粉样蛋白结构的第一次高分辨率一瞥提出了一些问题，这些问题似乎不能通过对淀粉样蛋白本身的研究来完全回答。因此，我们正在寻求一种基于淀粉样蛋白状态的可溶性模型的新方法。天然淀粉样蛋白和相关疾病之间的联系尚不清楚：毒性可能来自原纤维本身，低聚物前体，或两者兼而有之（目前大多数文献支持可溶性低聚物作为主要毒性剂）。无论病理效应的起源如何，对影响淀粉样蛋白状态的结构和稳定性的因素有一个基本的了解是至关重要的。由于淀粉样蛋白本身是不服从许多强大的战略可用于可溶性蛋白质的物理特性，我们寻求可溶性模型系统，表现出与真实的淀粉样蛋白结构的关键特征。单程跨膜α-螺旋（“SPTM螺旋”）之间的关联对于许多膜蛋白的功能至关重要。例如，双特异性受体含有将感觉性细胞外结构域与功能性细胞内结构域连接的单程螺旋。信号传导通常需要离散的受体组装体（二聚体、三聚体或更大的寡聚体）。形成的信号主管组件至少部分取决于SPTM螺旋之间的特定相互作用。近年来出现了基于NMR的SPTM螺旋组装体的结构模型，但据我们所知，还没有双特异性受体衍生的SPTM螺旋组装体的晶体学数据。唯一相关的晶体结构涉及流感M2质子通道的SPTM片段，其形成四聚体，以及来自DAP 12（一种免疫受体衔接蛋白）的TM螺旋的最近结构。我们的长期目标是获得多种晶体结构的不同SPTM螺旋组件，从而有助于共同阐明的规则，管理膜内螺旋螺旋识别。无规和准无规结晶是我们研究的主要工具。SPTM螺旋组件的晶体结构从双位受体将是非常重要的补充NMR为基础的模型。螺旋间界面的性质是最紧迫的问题，晶体结构将提供通过NMR可能无法获得的有价值的见解。

 DESCRIPTION (provided by applicant): 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 α-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.