Protein Structural Defects: Sites for Small Molecule Binding and Regulation

该项目将研究新的实验方法，以检测蛋白质用来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完成许多任务至关重要，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。了解这些辅因子是如何与蛋白质靶标结合的--关键是知道它们在蛋白质上的作用位置--对于理解正常的生物功能是如何实现的，这种功能在疾病中是如何受到干扰的，以及人们如何为生物技术或医学产生新的调节分子是至关重要的。该项目将研究开发一种新的方法来解决辅因子在哪里以及如何与蛋白质结合的问题，方法是利用加压水来帮助快速探测辅因子结合位点，并结合核磁共振光谱来获得原子精度。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，将结果应用于广泛的蛋白质靶标，并培训学生和研究人员结合尖端生物物理和生化方法和分析。该项目将多种溶液核磁共振方法与互补的实验和计算研究相结合，研究蛋白质中不寻常的空穴结构和动力学如何形成适合小分子结合和调节的位点。这项工作对于提供配体结合位点性质的定量描述，促进对生物学基本原理的理解，以及能够开发用于化学和合成生物界的新型基因编码传感器和配体激活蛋白具有重要意义。这项研究计划专注于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洞，其中许多在其核心中具有微小的水合缺陷，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目前仍不清楚，大多数都围绕着配体结合是如何发生的，尽管这些空腔与水完全隔离。此外，人们几乎不了解蛋白质动力学是如何参与这种蛋白质-配体相互作用的-众所周知，蛋白质动力学影响配体的专一性和变构变化在许多其他系统中的传递。为了解决这些缺点，该项目将利用高压溶液核磁共振波谱与其他生物物理和生化方法相结合，以：1)。确定一个结构域子集内的空洞患病率和配体结合的流行率，2)。确定高压如何影响腔结构和动力学，允许在有和没有配体和3)的情况下访问不同的构象。研究配体结合到控制效应域的功能连接。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.