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的基本兴趣，因为蛋白质不稳定性是多种病理的原因，它将使工业和治疗目的的蛋白质工程变得容易。蛋白质工程和从头设计广泛地描述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，我们离蛋白质稳定性的精确物理化学模型还很遥远；目前还没有可靠的方法来预测任意突变的热力学结果。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，通过构建大型的靶向蛋白质变体库并对这些变体进行折叠性排序。现代技术使得对折叠蛋白变异的统计意义显著的样本进行分类和排序，以探测对稳定性的微妙影响，变得既经济又实用。为了利用一次性从头设计的智慧，我们开发了体内和体外的方法来分类和分析一种已经研究得很好的模型蛋白，同二聚体四螺旋束Rop。目标文库将使用非常高通量的基于细胞的荧光屏幕和一种新颖的、中等高通量的疏水染料结合方法（高通量量热法）进行稳定性排序，该方法可以揭示详细的热力学信息。通过设计无半胱氨酸且活性良好的单链Rops，我们将扩展这些研究，以比较蛋白质界面的直接序列决定因素与小单体蛋白质的疏水核心。我们正在合作利用单分子光谱学来理解设计变体的构象平衡。四螺旋束包含许多治疗和病理学上有趣的蛋白质，但为了解决与人类疾病直接相关的模型的稳定性，以及将2-sheet蛋白与螺旋蛋白进行比较，我们正在开发类似的肿瘤抑制因子p53核心结构域的筛选技术。除了筛选与Rop核心文库类似的文库外，我们还将筛选和表征MD模拟预测的具有减少动态运动的p53变体。生物物理表征的通量普遍较差，导致大多数支架只对少数蛋白质突变体进行了详细的检测。这阻碍了对序列对比或较浅能量面勘探等影响的深入研究。在这里，我们将利用高通量方法的力量来测试和改进具有统计显著性的蛋白质设计原则，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.