Engineering of cold denaturing proteins

蛋白质是由数百或数千个氨基酸残基组成的大分子，这些氨基酸按一定的顺序排列形成聚合物。它们对所有生物都至关重要，对卫生保健和生物技术也至关重要。蛋白质分子表现出独特的三维结构特征，支配着它们的功能。然而，氨基酸序列如何决定结构特征还不完全清楚。特别是，这适用于“疏水效应”，这是因为一些具有“类油性质”的氨基酸在水中完全不溶。因此，这些残留物往往形成蛋白质分子的内部核心(就像水中的一个微小的油滴)，以最大限度地减少与周围水的接触。对疏水效应的不完全理解源于蛋白质-水相互作用的巨大复杂性。反过来，这又阻碍了从新蛋白质的序列和准确设计中准确预测结构和稳定性。该项目的一个主要潜在好处将是显著增强对疏水效应的理解。这将通过在新的综合方案中将尖端生物物理学与计算蛋白质设计方法相结合来实现，以实现更准确的蛋白质结构预测和工程设计。该项目的所有协议和产品都将公开提供。在蛋白质生物物理学和计算设计方面对年轻科学家，特别是来自代表人数较少的少数民族的青年科学家进行出色的培训，是该项目的一个主要目标。除了高度个人化的研究和职业发展指导外，还将举办一年一度的暑期学校，以接触生物物理研究以外的社区，并提高人们对“STEM”的普遍兴趣。蛋白质热力学的一个中心猜想是，蛋白质的结构状态以及在极低温度下(‘冷变性’)蛋白质结构的丧失是疏水效应的结果。这个项目将专注于冷变性蛋白质的工程设计，通过使用Rosetta程序在蛋白质结构和热力学曲线的计算设计和设计的生物物理特征之间进行迭代。热力学表征将通过基于圆二色谱(CD)的Gibbs-Helmholtz分析和差示扫描量热法(DSC)进行，结构和变性状态的结构表征将通过CD和核磁共振(NMR)光谱以及小角X射线散射(SAXS)进行。这种迭代的蛋白质工程将以前所未有的方式测试和提高我们对蛋白质热力学和计算设计方法的理解。

Proteins are large molecules consisting of hundreds or thousands of amino acid residues forming a polymer with the amino acids arranged in a defined sequence. They are pivotal for all living organisms and of central importance for health care and biotechnology. Protein molecules exhibit distinct three- dimensional structural features governing their function. However, it is not entirely clear how the amino acid sequence determines structural features. In particular, this holds for the "hydrophobic effect" which arises because some amino acids with 'oil like properties' are quite insoluble in water. As a result, these residues tend to form an interior core of the protein molecule (alike a tiny oil droplet in water) to minimize exposure to the surrounding water. The incomplete understanding of the hydrophobic effect results from the enormous complexity of protein-water interactions. In turn, this prevents accurate prediction of structure and stability from sequence and accurate design of novel proteins. A primary potential benefit of this project will be a significant enhancement of the understanding of the hydrophobic effect. This will be accomplished by combining cutting-edge biophysics with computational protein design methodology in new and integrated protocols to enable more accurate protein structure prediction and engineering. All protocols and products of this project will be made publicly available. Excellent training of young scientists, in particular from underrepresented minorities, in protein biophysics and computational design, is a major goal of this project. Apart from highly individual mentoring of research and career development, annual summer schools will be held to reach communities outside of biophysical research and to increase interest in "STEM" in general.It is a central conjecture of protein thermodynamics that the structured state of a protein as well as the loss of protein structure at very low temperatures ('cold denaturation') result from the hydrophobic effect. This project will focus on the engineering of cold denaturing proteins by iterating between computational design of protein structure and thermodynamic profile using the program Rosetta and biophysical characterization of the designs. The thermodynamic characterization will be pursued by use of circular dichroism (CD)-based Gibbs-Helmholtz analyses as well as differential scanning calorimetry (DSC), and the structural characterization of structured and denatured states by use of CD and nuclear magnetic resonance (NMR) spectroscopy, and small angle X-ray scattering (SAXS). Such iterative protein engineering will test and improve in an unprecedented manner our understanding of protein thermodynamics and computational design methodology.