Protein Structural Defects: Sites for Small Molecule Binding and Regulation

该项目将研究新的实验方法来检测蛋白质用于结合对其功能至关重要的小分子辅因子的位点。 这些知识对于理解细胞如何使用基于蛋白质的分子机器执行许多任务至关重要，从感知微环境的变化到控制内部复杂生化反应的速度。 这些蛋白质机器通常利用小分子辅因子来执行和控制它们的各种功能。 了解这些辅因子如何结合其蛋白质靶点--以及关键的是，它们在蛋白质上的何处结合--对于理解正常生物功能是如何实现的、这种功能在疾病中是如何被扰乱的以及如何为生物技术或医学产生新的调节分子至关重要。 该项目将研究一种新方法的开发，通过利用加压水来帮助快速探测辅因子结合位点，再加上NMR光谱来获得原子精度，来解决辅因子在哪里以及如何与蛋白质结合。 计划研究的成功完成将产生可广泛应用于学术和工业实验室的新技术，应用于广泛的蛋白质靶点，以及对学生和研究人员的培训，边缘生物物理和生物化学的方法和分析。该项目结合了多个解决方案核磁共振方法与互补的实验和计算研究，以调查如何不寻常的蛋白质内空腔的结构和动力学形成适合于小分子结合和调节的位点。 这些工作在提供配体结合位点性质的定量描述，推进对生物学基本原理的理解以及开发可用于化学和合成生物学社区的新型遗传编码传感器和配体活化蛋白方面具有重要意义。 该研究计划的重点是研究配体控制的蛋白质/蛋白质相互作用结构域大家族中的空腔，其中许多在其核心内具有小的水合缺陷，这些缺陷可能作为调节小分子结合位点。 这种结合的几个基本方面在这个时候仍然是神秘的，大多数围绕着配体结合如何发生，尽管这些空腔完全与水隔离。 同样，几乎没有人了解蛋白质动力学-众所周知影响配体特异性和许多其他系统中变构变化的传递-如何参与这种蛋白质-配体相互作用。 为了解决这些缺点，该项目将利用高压溶液NMR光谱与其他生物物理和生物化学方法相结合：1）。确定空腔流行率和配体结合在一个域的子集内的流行率，2）。确定高压如何影响空腔结构和动力学，允许在有和没有配体的情况下访问不同的构象; 3）。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

This project will investigate novel experimental approaches to detect sites used by proteins to bind small molecule cofactors that are essential to their function. Such knowledge is critical to understanding how cells use protein-based molecular machines for many tasks, ranging from sensing changes in their microenvironment to controlling the speed of complex biochemical reactions inside of them. These protein machines often utilize small molecule cofactors to perform and control their various functions. Knowing how these cofactors bind their protein targets - and critically, where they do so on the protein - is essential for understanding how normal biological function is achieved, how this function is perturbed in disease, and how one might generate new regulatory molecules for biotechnology or medicine. This project will investigate the development of a novel approach to address where and how cofactors bind to proteins by taking advantage of pressurized water to help quickly probe for cofactor-binding sites, coupled with NMR spectroscopy to yield atomic precision. The successful completion of the planned research will yield new techniques which can be widely applied in academic and industrial laboratories, application results to a wide range of protein targets, and the training of students and researchers in a combination of cutting-edge biophysical and biochemical methods and analyses.This project combines multiple solution NMR approaches with complementary experimental and computational studies to investigate how the unusual structure and dynamics of cavities within proteins form sites suitable for small molecule binding and regulation. Such work is significant in providing quantitative descriptions of ligand-binding site properties, advancing the understanding of fundamental principles in biology and enabling the development of novel genetically-encoded sensors and ligand-activated proteins useful for the chemical and synthetic biology communities. This research program focuses on studying cavities within a large family of ligand-controlled protein/protein interaction domains, many of which have small, hydrated defects within their cores which can potentially serve as regulatory small-molecule binding sites. Several fundamental aspects of such binding remain cryptic at this time, most surrounding how ligand binding can occur despite these cavities being completely isolated from water. As well, there is almost no understanding of how protein dynamics - well-known to impact ligand specificity and the transmission of allosteric changes in many other systems - are involved in such protein-ligand interactions. To address these shortcomings, this project will utilize high pressure solution NMR spectroscopy in combination with other biophysical and biochemical approaches to: 1). Ascertain the prevalence of cavity prevalence and ligand binding within a subset of domains, 2). Determine how high pressure affects cavity structure and dynamics, allowing different conformations to be accessed with and without ligands and 3). Examine the functional linkage of ligand binding to control of effector domains.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.