Collaborative Research: Energy Landscapes of Designed Cold Unfolding Proteins

蛋白质是在活的有机体中通过连接数百甚至数千个氨基酸形成线性链而合成的非常大的生物分子。蛋白质是生命所必需的，因此它们是基础和生物医学研究的重要目标，包括药物开发和生物技术。催化生物化学反应的酶、蛋白质药物如胰岛素和传递免疫力的抗体是众所周知的例子。重要的是，蛋白质的生物学功能与线性链可以采用的许多不同的三维形状密切相关，这些形状严重依赖于温度和压力。该项目的目的是加强对蛋白质在极端条件下，即在水的冰点以下的极低温度和/或在数百个大气压的极高压力下所采取的独特形状的理解。这需要了解在这些条件下存在的巨大生态系统中的生命（例如在海洋和极地地区），了解这些形状如何与蛋白质功能，蛋白质相关疾病，蛋白质疫苗开发和蛋白质药物制剂相关，并加强生物技术和生物医学重要的“冷适应蛋白”的工程。这项研究有助于制定规则来预测蛋白质的形状及其在这种极端条件下的能量特性，新开发的方法和计算工具将提供给更广泛的科学界。该项目能够对研究人员，包括代表性不足群体的科学家进行跨学科培训。为了实现这一目标，并提高对STEM的兴趣，Kuhlman博士和Szyperski博士在他们的学校参加了完善的重大举措，这些举措致力于促进包容性社区，留住STEM中代表性不足的学生，并指导学生。他们还参与NSF资助的本科生研究机会，并提供关于“关键”的网络研讨会，以了解（生物）物理化学和（生物）物理学。此外，还将举办程序设计工作坊，重点介绍分子建模方法。蛋白质在不同温度/压力下的不同三维分子形状之间的关系与其分子能量和熵有关，这些能量和熵由所谓的“能量熵”表示。在低温下趋于失去明确形状的蛋白质被称为“冷解折叠蛋白质”。研究的重点是探索和理解这些蛋白质的能量景观。这包括“冷”低能量/低熵状态的结构和热力学性质，它们向其他状态的转变，以及对蛋白质压力-温度“相变”的更深入理解。采用结合从头计算蛋白质设计、尖端生物物理技术和分子动力学（MD）模拟的独特方法，以专门测试中心结构和热力学假设，即（i）混合的疏水/亲水蛋白质核心导致部分冷的展开冷状态，其中水分子形成不可分割的部分，（ii）这表现在复杂的能量景观中，（iii）这导致形成这种冷态的明显的热力学特征，以及（iv）冷态形成的协同性随着折叠芯的亲水含量的增加而降低。此外，这项研究为解决冷态可能在功能上很重要的假设奠定了基础，即使在人口稀少的环境条件下。最后，开发了新的计算设计方案，其促进或使得能够设计含有冷解折叠蛋白质的β-折叠，以及将折叠的天然存在的蛋白质重新设计为在极端条件下解折叠的同源物，以验证新建立的原理。该项目由分子和细胞生物科学部的分子生物物理学小组支持。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteins are very large biological molecules synthesized in living organisms by linking hundreds or even thousands of amino acids to form a linear chain. Proteins are required for life and they are thus important targets for fundamental and biomedical research, including drug development, and biotechnology. Enzymes catalyzing biochemical reactions, protein drugs such as insulin, and antibodies conveying immunity are well-known examples. Importantly, the biological function of proteins is intimately linked to the many different three-dimensional shapes the linear chains can adopt, and these shapes depend critically on both temperature and pressure. The objective of this project is to enhance the understanding of the distinct shapes which proteins adopt at extreme conditions, that is, at very low temperatures below the freezing point of water and/or at very high pressures of several hundreds of atmospheres. This is required to understand life in the vast ecosystems existing under these conditions (e.g. in oceans and the polar regions), to understand how these shapes relate to protein function, protein related diseases, protein vaccine development, and protein drug formulation, and to enhance the engineering of bio-technologically and bio-medically important ‘cold adapted proteins’. The research contributes to develop rules to predict protein shapes and their energetic properties under such extreme conditions, and newly developed methodologies and computational tools will be made available to the broader scientific community. The project enables cross-interdisciplinary training of researchers, including scientists from underrepresented groups. To pursue this goal and to increase interest in STEM in general, Drs. Kuhlman and Szyperski, participate in well-established, major initiatives at their schools which are dedicated to promote inclusive communities, to retain underrepresented students in STEM and to mentor students. They also participate in NSF-funded research opportunities for undergraduates, and offer webinars on ‘linchpins’ for the understanding of (bio)physical chemistry and (bio)physics. Programming workshops focusing on methods for molecular modeling will also be offered.The relationship between the different three-dimensional molecular shapes of proteins dominating at different temperatures / pressures is related to their molecular energies and entropies, which are represented by so called ‘energy landscapes’. Proteins which tend to lose a well-defined shape at low temperatures have been named ‘cold unfolding proteins’. Research focuses on exploring and understanding the energy landscapes of such proteins. This includes the structural and thermodynamic properties of the ‘cold’ low energy / low entropy states, their transitions to other states and, in general, a more advanced understanding of protein pressure-temperature ‘phase diagrams’. A unique approach combining computational de novo protein design, cutting-edge biophysical techniques and molecular dynamics (MD) simulations is employed in order to specifically test central structural and thermodynamic hypotheses, namely that (i) a mixed hydrophobic / hydrophilic protein core results in a partially cold unfolded cold state in which water molecules form an integral part, (ii) this is manifested in complex energy landscapes, (iii) this results in a distinct thermodynamic signature for the formation of such cold states, and (iv) the co-operativity of the formation of the cold states decreases with an increasing hydrophilic content of the folded core. Moreover, the research lays the foundation to tackle the hypothesis that cold states can be functionally important, even at ambient conditions when they are lowly populated. Finally, new computational design protocols are developed which facilitate or enable the design of beta-sheet containing cold unfolding proteins, and the redesign of folded, naturally occurring proteins to congeners which unfold under extreme conditions in order to validate newly established principles. This project is supported by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.