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

蛋白质是生命系统的分子机器，就像任何机器一样，依靠运动来实现它们的功能。对于存在于定义细胞边界的脂膜中的蛋白质也是如此，称为膜蛋白。一类经历运动的膜蛋白似乎对功能很重要，这类蛋白是膜内蛋白酶的菱形家族。在所有生命王国中都发现了菱形动物，它们在广泛的生物过程中发挥着作用，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目标。然而，我们控制菱形功能的能力取决于对菱形如何能够唯一地切割靶蛋白的理解，这些靶蛋白通常嵌入膜的缺水环境中，处于一种抑制需要水的反应的状态。有大量证据表明，菱形和它的靶子都必须采用一种以上的结构才能实现这种转变，这可能是催化循环中最慢的步骤，从而控制菱形的活性。然而，这种构象变化的性质，以及这些动态如何被脂质环境调节尚不清楚，这代表了我们对菱形活性理解的一个关键差距。我们将使用细菌中一种特性良好的菱形蛋白酶作为我们的模型系统，称为GlpG，研究菱形功能与其动力学之间的关系，以及膜环境在调节种群和交换率中的作用。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，这些信号报告了结构、能量和构象交换速率。由于GlpG是一种大的完整膜蛋白，因此有必要使用通常为大蛋白的核磁共振研究而保留的特殊策略。这将包括通过选择性标记方案引入独特的核磁共振活性标记、特定部位的化学标记和遗传密码扩展以引入具有所需标记的非天然氨基酸。后一种策略特别令人兴奋，因为它将首次为研究天然细菌膜中的GlpG动力学打开大门。我们将分离含有选择性标记的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。这一新方法有可能应用于研究其他天然膜环境中的动态膜蛋白。综上所述，这个项目的结果将提高我们对动力学如何调节菱形蛋白酶功能，脂膜性质如何调节这些动力学，以及我们如何最终能够控制动力学来改变功能的理解。

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.