Combinatorial biophysics: understanding protein stability with library approaches

描述（由申请人提供）：对序列-稳定性关系的精确理解是蛋白质生物化学中的基本兴趣，因为蛋白质不稳定性是多种病理学的原因，并且它将使得能够容易地工程化蛋白质用于工业和治疗目的。蛋白质工程和从头设计已经大致描绘了稳定蛋白质的力量，并在设计新的或稳定的蛋白质方面取得了一些惊人的成功。然而，我们仍然远离蛋白质稳定性的精确物理化学模型;仍然没有可靠的方法来预测任意突变的热力学后果。在这里，我们提出了通过从头蛋白质设计开发的假设的直接测试，通过构建大的，有针对性的蛋白质变体库，并对这些变体进行折叠排序。现代技术使得对折叠蛋白质变体的重要样品进行分类和测序以探测对稳定性的微妙影响变得经济实惠和实用。为了充分利用一次一个从头设计的智慧，我们已经开发了体内和体外方法，用于分选和测定一个经过充分研究的模型蛋白质，同源二聚体四螺旋束Rop。靶向文库将使用非常高通量的基于细胞的荧光筛选和揭示详细热力学信息的新型中等高通量疏水染料结合方法（高释放量热法）进行稳定性分选。通过工程化无半胱氨酸和活性的，表现良好的单链Rops，我们将扩展这些研究，直接比较蛋白质-蛋白质界面的序列决定簇与小单体蛋白质的疏水核心。我们正在合作，以了解使用单分子光谱设计的变体的构象平衡。四螺旋束包括许多治疗和病理学上感兴趣的蛋白质，但为了解决与人类疾病直接相关的模型中的稳定性，以及将2片层蛋白质与螺旋蛋白质进行比较，我们正在开发肿瘤抑制因子p53的核心结构域的类似筛选技术。除了筛选类似于Rop核心文库的文库外，我们还将筛选和表征从MD模拟预测的具有减少的动态运动的p53变体。生物物理表征的通量通常很差，结果是对于大多数支架，只有少数蛋白质突变体被详细检查。这阻碍了对序列相关或较浅能量表面勘探等效应的彻底研究。在这里，我们将使用高通量方法的力量来测试和改进具有统计学意义的蛋白质设计原则，既提高了我们对序列结构关系的认识，又使未来的设计和治疗方法成为可能。

DESCRIPTION (provided by applicant): A precise understanding of the sequence-stability relationship is of fundamental interest in protein biochemistry, as protein instability is a cause of a wide range of pathologies, and it would enable facile engineering of proteins for industrial and therapeutic purposes. Protein engineering and de novo design have broadly delineated the forces that stabilize proteins and have yielded some spectacular successes in designing new or stabilized proteins. However, we are still far from a precise physicochemical model of protein stability; there is still no reliable way to predict the thermodynamic consequences of an arbitrary mutation. Here, we propose direct tests of the hypotheses that have been developed through de novo protein design, by building large, targeted libraries of protein variants and sorting those variants for foldedness. Modern technology makes it affordable and practical to sort and sequence a statistically-significant sample of folded protein variants to probe subtle effects on stability. To leverage the wisdom of one-at-a-time de novo design, we have developed in vivo and in vitro methods for sorting and assaying a well-studied model protein, the homodimeric four-helix bundle Rop. Targeted libraries will be sorted for stability using a very high throughput cell-based fluorescence screen, and a novel, moderately high-throughput hydrophobic dye binding method (High Throughput Calorimetry) that reveals detailed thermodynamic information. By engineering cysteine-free and active, well-behaved single-chain Rops, we will expand these studies to compare directly sequence determinants of protein-protein interfaces versus hydrophobic cores of small, monomeric proteins. We are collaborating to understand the conformational equilibria of designed variants using single-molecule spectroscopy. Four-helix bundles comprise many therapeutically and pathologically interesting proteins, but to address stability in a model directly relevant to human disease, as well as to compare a 2-sheet protein to a helical one, we are developing analogous screening technology for the core domain of the tumor suppressor p53. In addition to screening libraries analogous to the Rop core libraries, we will screen and characterize p53 variants predicted from MD simulation to have reduced dynamic motions. The throughput of biophysical characterization is generally poor, and the result is that only a small number of protein mutants have been examined in detail for most scaffolds. This prevents thorough study of effects such as sequence correlation or exploration of shallower energy surfaces. Here, we will use the power of high- throughput approaches to test and refine protein design principles with statistical significance, both improving our knowledge of the sequence-structure relationship and enabling future design and therapeutic approaches.