NIH DIRECTOR'S PIONEER AWARD

令人惊讶的是，人们对蛋白质在体内如何折叠知之甚少，但这是这个过程，而不是试管 理想化折叠反应在过去几十年中得到了如此深入的研究，这对 生物体的适应性。折叠的保真度和细胞中蛋白质的稳定性对它们的 函数、它们的退化以及它们对聚合的脆弱性。许多疾病现已为人所知 由于必需蛋白质的丢失或改变而引起的蛋白质折叠缺陷 功能，或由于聚集体等有毒物种的积聚。我们相信那本小说 方法和创造性合作将使我们能够克服令人望而生畏的技术障碍， 阻碍了细胞内蛋白质折叠的进展。首先关注的是一种小的蛋白质模型， 我们有详细的体外折叠的描述，将使方法优化。在细胞中折叠 在日益复杂的系统中，将遵循以下条件：细菌蛋白表达，无细胞 生物合成，半透性或完整的真核细胞表达。新战略将 揭示生物医学感兴趣的较大蛋白质如何在细胞环境中采用它们的结构，以及如何 这一过程可能会出错。在方法论上，我们预计主要依靠光谱学 方法，包括荧光和核磁共振，并使用新的标记策略 在复杂的细胞环境中观察正在研究的蛋白质。互补细胞内成像 将使用方法来确保观察到的信号报告相关现象并揭示新的 功能上重要的空间定位模式。我们预计这项研究将带来新的 氨基酸序列如何编码折叠信息以及由此产生的增强的 对体内折叠的了解将为错误折叠的治疗干预和 聚集性疾病。

Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube idealized folding reaction so intensively studied over the past several decades, that is crucial to the fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to arise from defects in protein folding, either because of the loss or alteration of essential protein functions, or because of the build-up of toxic species such as aggregates. We believe that novel methods and creative collaborations will allow us to overcome the daunting technical obstacles that have impeded progress on protein folding in the cell. Focusing first on a small model protein for which we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging methods will be used to insure that observed signals report on relevant phenomena and to reveal novel functionally significant spatial localization patterns. We anticipate that this research will lead to new paradigms for how amino acid sequences encode folding information and that the resulting enhanced understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and aggregation diseases.