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，依靠运动来实现它们的功能。对于存在于定义细胞边界的脂质膜上的蛋白质也是如此，称为膜蛋白。一类膜蛋白的运动似乎是重要的功能是菱形家族的膜内蛋白酶。菱形体存在于所有生命领域，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，人们对开发能够抑制或激活菱形蛋白酶的化合物感兴趣，使其成为药物发现的目标。然而，我们控制菱形功能的能力依赖于了解菱形是如何独特地切割目标蛋白的，这些蛋白通常嵌入在膜的缺水环境中，从而抑制需要水的反应。有大量证据表明，菱形和它的目标必须采用不止一种结构来允许这种转变，这可能是催化循环中最慢的一步，从而控制菱形活性的速率。然而，这种构象变化的性质以及这些动力学如何被脂质环境调节尚不清楚，这代表了我们对菱形活性的理解中的一个关键空白。利用一种从细菌中提取的具有良好特征的菱形蛋白酶作为我们的模型系统，称为GlpG，我们将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速率中的作用。我们将利用固相核磁共振（NMR）的能力来解析来自单个原子的信号，这些信号可以报告结构、能量学和构象交换速率。由于GlpG是一种大的整体膜蛋白，因此有必要使用通常用于大蛋白核磁共振研究的特殊策略。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，位点特异性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。后一种策略尤其令人兴奋，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GlpG的天然膜的稳定片段，并研究其在天然环境中的菱形动力学。这种新方法有可能应用于其他动态膜蛋白在其原生膜环境中的研究。总的来说，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的理解，脂质膜特性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。

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.