Determinants of Fold Stability in Proteins and Analogues

描述(申请人提供)：我们的研究旨在阐明蛋白质所显示的特定折叠和组装行为的起源。蛋白质具有多种生命所必需的功能，蛋白质的活性通常依赖于多肽链对高阶结构的采用。因此，了解控制折叠和组装的因素，即二级结构、三级结构和通常是四级结构的形成，对于给定的氨基酸残基序列是一个基本的科学目标。我们的研究使用了标准的表征工具；此外，我们还努力创建新的工具，为重要问题提供独特的方法。例如，我们正在开发一组新的分子单元，使平行β-折叠二级结构的热力学分析成为可能，目前的一个目标是使用这些工具来阐明基本参数，如链的数量或链的长度如何影响平行β-折叠的稳定性。在这种情况下，我们独特的工具是非天然的二胺或二酸片段，它们通过C-或N-末端连接多肽片段，并促进水中平行的β-折叠的形成。这样的连接物在经典的蛋白质科学中是不存在的，这项研究需要一个像我们这样的实验室，在生物物理表征和有机合成方面都有经验。我们团队最近开发的另一种化学工具，“主链硫酯交换”(BTE)方法，提供了一种独特的方法来分析小分子多肽在二级、三级和/或四级结构水平上的序列稳定性相关性。拟议的研究包括使用BTE来探索在α-螺旋片段(例如，盘绕线圈)之间的并排相互作用中控制亲和力和选择性的因素。螺旋-螺旋缔合是蛋白质三级和四级结构的一个显著特征，我们的研究将解决这一领域以前未回答的问题。此外，我们还建议将BTE扩展到脂双层中螺旋-螺旋相互作用的研究。与已知的溶液中蛋白质相比，目前对控制蛋白质在膜上折叠和结合的作用力的了解还不够深入，膜蛋白质领域的一个主要障碍是缺乏有效的结构现象的热力学分析方法。BTE可以代表这一领域的一个强大的新工具。我们希望将我们的理解从蛋白质扩展到显示出蛋白质样结构的非天然低聚物。这些努力将扩大对非共价相互作用网络控制柔性低聚物构象和结合倾向的方式的基本理解。此外，我们研究的这一部分是基于这样的长期前景，即具有众所周知的折叠规则的非天然低聚物可以为创造新型生物医学有用的制剂提供基础。与公共健康相关的蛋白质是生命的主力分子。蛋白质的功能在很大程度上取决于它们所采用的结构，我们工作的一个主要目标是阐明控制蛋白质结构的因素。表现出蛋白质样结构行为的非天然分子最终可能被设计成具有生物医学有用的蛋白质样功能，我们的目标包括发现和表征新的蛋白质模拟系统。

DESCRIPTION (provided by applicant): Our research is intended to elucidate the origins of specific folding and assembly behavior displayed by proteins. Proteins perform a vast array of functions that are essential for life, and protein activity usually depends on the adoption of higher order structure by the polypeptide chain. Therefore, understanding the factors that control folding and assembly, i.e., secondary, tertiary and often quaternary structure formation, for a given sequence of amino acid residues is a fundamental scientific goal. Our research employs standard characterization tools; in addition, we strive to create new tools that offer unique approaches to important questions. For example, we are developing a set of novel molecular units that enable thermodynamic analysis of parallel beta-sheet secondary structure, and one current goal is to use these tools to elucidate how basic parameters such as number of strands or length of strands influence parallel beta-sheet stability. Our unique tools in this case are unnatural diamine or diacid segments that link peptide segments via their C- or N-termini and promote parallel beta-sheet formation in water. Such linkers are not available in classical protein science, and this research requires a laboratory, such as ours, that has experience in both biophysical characterization and organic synthesis. Another chemical tool recently developed by our group, the "backbone thioester exchange" (BTE) method, offers a unique approach to analysis of sequence-stability correlations at secondary, tertiary and/or quaternary structure levels in small polypeptides. The proposed research includes the use of BTE to probe the factors that govern affinity and selectivity in side-by-side interactions between alpha-helical segments (e.g., coiled-coils). Helix-helix association is a prominent feature of protein tertiary and quaternary structure, and our studies will address previously unanswered questions in this area. In addition, we propose to extend BTE to the study of helix-helix interactions in lipid bilayers. Current understanding of the forces that control protein folding and association in membranes is underdeveloped relative to what is known about proteins in solution, and a major stumbling block in the membrane protein field is lack of effective methods for thermodynamic analysis of structural phenomena. BTE could represent a powerful new tool in this field. We want to extend our understanding beyond proteins to unnatural oligomers that display protein-like structures. Such efforts will broaden fundamental understanding of the ways in which networks of noncovalent interactions control the conformations and binding propensities of flexible oligomers. In addition, this component of our research is motivated by the long-term prospect that unnatural oligomers with well-understood folding rules could provide a basis for creating new kinds of biomedically useful agents. PUBLIC HEALTH RELEVANCE Proteins are the workhorse molecules of life. The functions of proteins depend critically on the structures they adopt, and a major goal of our work is to elucidate factors that govern protein structure. Unnatural molecules that display protein-like structural behavior could ultimately be engineered to display biomedically useful protein-like functions, and our goals include the discovery and characterization of new protein-mimetic systems.