Collaborative Research: Energy Landscapes of Designed Cold Unfolding Proteins

蛋白质是在生物体中通过连接数百甚至数千个氨基酸形成线性链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。催化生化反应的酶、胰岛素等蛋白质药物和传递免疫力的抗体就是众所周知的例子。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，而这些形状严重依赖于温度和压力。该项目的目标是加强对蛋白质在极端条件下的不同形状的理解，即在低于水冰点的极低温度和/或在数百个大气压的非常高的压力下。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配方的关系，以及加强生物技术和生物医学上重要的“冷适应蛋白质”的工程设计，都需要这样做。这项研究有助于开发预测蛋白质形状及其在这种极端条件下的能量特性的规则，并且新开发的方法和计算工具将为更广泛的科学界提供。该项目能够对研究人员进行跨学科培训，包括来自代表性不足群体的科学家。为了实现这一目标，并提高人们对STEM的普遍兴趣。Kuhlman和Szyperski在他们的学校参与了一些完善的重大倡议，这些倡议致力于促进包容性社区，保留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，并提供关于理解（生物）物理化学和（生物）物理的“关键”的网络研讨会。还将提供以分子建模方法为重点的编程研讨会。在不同温度/压力下占主导地位的蛋白质的不同三维分子形状之间的关系与它们的分子能量和熵有关，这由所谓的“能量景观”表示。在低温下容易失去明确形状的蛋白质被称为“冷展开蛋白质”。研究的重点是探索和理解这类蛋白质的能量格局。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“相图”的更深入理解。一种独特的方法结合了计算从头蛋白质设计，尖端的生物物理技术和分子动力学（MD）模拟，以专门测试中心结构和热力学假设，即(i)混合疏水/亲水蛋白质核心导致部分冷展开冷状态，其中水分子是不可分割的一部分，（ii）这在复杂的能量景观中表现出来。(3)这导致了冷态形成的明显热力学特征，(4)随着折叠核亲水性含量的增加，冷态形成的协同性降低。此外，该研究为解决冷态可能具有重要功能的假设奠定了基础，即使在人口稀少的环境条件下也是如此。最后，开发了新的计算设计协议，以促进或实现含有冷展开蛋白质的β片的设计，并重新设计折叠的，自然发生的蛋白质在极端条件下展开的同源物，以验证新建立的原则。本项目由分子与细胞生物科学部分子生物物理组支持。该奖项反映了美国国家科学基金会的法定使命，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.