Combinatorial biophysics: understanding protein stability with library approaches

描述(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，这将使用于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引人注目的成功。然而，我们距离蛋白质稳定性的精确物理化学模型还很远；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，我们建议对通过从头蛋白质设计开发的假设进行直接测试，方法是建立大型的、有针对性的蛋白质变异库，并对这些变异体进行折叠排序。现代技术使得对具有统计学意义的折叠蛋白质变体样本进行排序和测序变得负担得起和实用，以探索对稳定性的微妙影响。为了利用一次一个的从头设计的智慧，我们开发了体内和体外的方法来分类和分析一个研究得很好的模型蛋白质，同源二聚体四螺旋束状Rop。目标文库将使用非常高吞吐量的基于细胞的荧光屏和揭示详细热力学信息的新型、适度高通量疏水染料结合方法(高通量热量计)进行稳定性筛选。通过设计无半胱氨酸和活性的、行为良好的单链Rop，我们将扩大这些研究，以直接比较蛋白质-蛋白质界面的序列决定因素与小分子单体蛋白质的疏水核心。我们正在合作，使用单分子光谱学来了解设计的变体的构象平衡。四螺旋束包含许多治疗和病理上感兴趣的蛋白质，但为了解决与人类疾病直接相关的模型中的稳定性问题，以及比较两页蛋白质和螺旋蛋白质，我们正在开发针对肿瘤抑制因子P53核心域的类似筛选技术。除了筛选与Rop核心文库类似的文库外，我们还将筛选和鉴定从MD模拟预测的具有减少动态运动的p53变体。生物物理表征的吞吐量通常很低，结果是只有一小部分蛋白质突变体被详细研究过。这妨碍了对层序对比或较浅能面探索等影响的深入研究。在这里，我们将使用高通量方法的力量来测试和提炼具有统计学意义的蛋白质设计原理，既提高了我们对序列-结构关系的了解，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.