Dynamic processes involved in intramembrane protease activity investigated by solution NMR.

蛋白质是生命系统的分子机器，像任何机器一样，依靠运动来执行其功能。这也适用于存在于定义细胞边界的脂质膜中的蛋白质，称为膜蛋白。一类经历似乎对功能重要的运动的膜蛋白是膜内蛋白酶的菱形家族。在所有生命王国中发现，菱形在广泛的生物过程中发挥作用，包括寄生虫感染，细胞生长和蛋白质质量控制。因此，人们有兴趣开发能够抑制或激活菱形蛋白酶的化合物，使其成为药物发现的靶点。然而，我们控制菱形功能的能力依赖于理解菱形如何能够独特地切割通常嵌入在膜的贫水环境中的靶蛋白，该靶蛋白处于抑制需要水的反应的状态。有大量的证据表明，菱形和它的目标必须采用一个以上的结构，以允许这种转变，这可能是催化循环中最慢的步骤，从而控制菱形活动的速率。然而，这种构象变化的性质，以及这些动力学如何可以调制的脂质环境是未知的，并代表了一个关键的差距，在我们的理解菱形活动。使用一个很好的特点菱形蛋白酶从细菌作为我们的模型系统，称为GlpG，我们将研究菱形功能和它的动力学之间的关系，以及在调节人口和交换率的膜环境的作用。我们将利用溶液相核磁共振（NMR）的能力来解析来自单个原子的信号，这些信号报告了构象交换的结构，能量和速率。由于GlpG是一个大的膜蛋白，它是必要的，使用特殊的策略通常保留的NMR研究的大蛋白质。这将包括通过选择性标记方案、位点特异性化学标记和遗传密码扩展引入独特的NMR活性标记，以引入具有所需标记的非天然氨基酸。后者的策略是特别令人兴奋的，因为它将首次打开大门，研究天然细菌膜中的GlpG动力学。我们将分离出含有选择性标记GlpG的天然膜的稳定片段，并研究其天然环境中的菱形动力学。这种新的方法有可能被应用到其他动态膜蛋白在其天然膜环境的研究。总之，该计划的结果将提高我们对动力学如何调节菱形蛋白酶功能，脂质膜特性如何调节这些动力学以及我们如何最终控制动力学以改变功能的理解。

Proteins are the molecular machines of living systems, and like any machine, rely on motions to carry out their functions. This is also true for proteins that exist in the lipid membrane that defines cellular boundaries, known as membrane proteins. One class of membrane proteins that undergo motions that appear to be important for function is the rhomboid family of intramembrane proteases. Found across all kingdoms of life, rhomboids play a role in a wide range of biological processes, including parasite infections, cell growth and protein quality control. Consequently, there is interest in developing compounds that can inhibit or activate the rhomboid protease, making them targets for drug discovery. However, our ability to control rhomboid function relies on understanding how the rhomboid can be uniquely capable of cleaving target proteins that are normally embedded within the water-poor environment of the membrane in a state that would be inhibitory against a reaction that requires water. There is a body of evidence suggesting that both the rhomboid and its target must adopt more than one structure to allow this transition, and that this may be the slowest step in the catalytic cycle, thereby controlling the rate of rhomboid activity. However, the nature of this conformational change, and how these dynamics can be modulated by the lipid environment is not known, and represents a key gap in our understanding of rhomboid activity. Using a well-characterized rhomboid protease from bacteria as our model system, called GlpG, we will study the relationship between rhomboid function and its dynamics, and the role of the membrane environment in modulating populations and rates of exchange. We will take advantage of the ability of solution phase nuclear magnetic resonance (NMR) to resolve signals from individual atoms that report on structure, energetics and rates of conformational exchange. Since GlpG is a large integral membrane protein it is necessary to use special strategies normally reserved for NMR studies of large proteins. This will include the introduction of unique NMR-active labels by selective labeling schemes, site-specific chemical labeling, and genetic code expansion to introduce unnatural amino acids having the desired label. This latter strategy is particularly exciting since it will for the first time open the door to the study of GlpG dynamics in native bacterial membranes. We will isolate stable fragments of native membrane containing selectively labeled GlpG and study rhomboid dynamics in its native environment. This new method has the potential to be applied to the study of other dynamic membrane proteins in their native membrane environments. Taken together, the results from this program will improve our understanding of how dynamics regulates rhomboid protease function, how lipid membrane properties can modulate these dynamics, and how we might be able to ultimately control dynamics to alter function.