CAREER: Proteins under Confinement: Revealing the Impact of Spatial Restrictions on Enzyme Structure, Dynamics and Function

蛋白质是许多细胞和生命过程中必不可少的大分子。在细胞中，蛋白质经历一个高度拥挤和受限的环境。因此，了解蛋白质在拥挤和受限条件下的结构、动力学和功能对于理解自然界中这些基本大分子的功能至关重要。这个项目将是一个最近开发的平台，它可以创建类似于典型蛋白质大小的受限空间，并使用现代光谱方法来检查蛋白质的结构和动力学。这些研究将被用来系统地理解空间限制如何影响蛋白质结构-功能关系。该项目将通过北达科他州州立大学和联合部落技术学院之间的暑期学生研究交流计划，改善北达科他州下一代科学家的沟通和合作。当地的本科生和高中研究人员也将被招募参与该项目。最后，该项目将激发当地小学生对科学的兴趣。该项目将确定空间限制的大小、形状和边界属性对蛋白质结构、动力学和功能的影响。该项目将使用最近开发的MOF/COF来系统地创建分别在大小、形状和边界属性上不同的受限空间。分别对溶菌酶和人铜锌超氧化物歧化酶(SOD1)两种模型蛋白的负载条件进行了优化，以优化其负载能力。溶菌酶和SOD1的活性将使用稍加修改的标准分析方法进行测定。然后将利用定点定向自旋标记将自旋探针植入每个模型蛋白质中。然后进行EPR动力学和远程分布测量，以确定两个模型蛋白质在不同MOF/COF中的全局和局部(活性部位和/或底物途径)构象柔性的变化。SDSL-EPR是这些实验的理想选择，因为它可以克服MOF/COF背景和蛋白质-MOF/COF相互作用造成的复杂性，并揭示所需的结构信息。在不同的限制条件下，将功能与结构细节相关联，将建立限制因素与蛋白质结构-功能关系之间的关联。该项目由分子和细胞生物科学部分子生物物理学分部(MCB)和既定的激励竞争研究计划(EPSCoR)共同资助。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。

Proteins are essential macromolecules for many cellular and life processes. In cells, proteins experience a highly crowded and confined environment. Understanding protein structure, dynamics, and function under crowding and confinement is, therefore, critical for understanding the function of these essential macromolecules in nature. This project will a recently developed platform that creates confined space at sizes similar to typical proteins, and use modern spectroscopic methods to examine protein structure and dynamics. These studies will be used to generate a systematic understanding on how spatial confinement impacts the protein structure-function relationship. This project will improve the communication and collaboration of next generation scientists in North Dakota by through a summer student research exchange program between North Dakota State University and the United Tribes Technical College. Local undergraduate and high school researchers will also be recruited to participate in the project. Lastly, the project will stimulate the interest in science for local elementary school students.This project will determine the influence of the size, shape, and boundary properties of spatial confinement on protein structure, dynamics, and function. This project will employ the recently developed MOFs/COFs to create systematically confined spaces that differ in size, shape, and boundary properties, respectively. The loading conditions of two model proteins, lysozyme and the human Cu/Zn superoxide dismutase (SOD1), will be optimized separately in order to optimize their loading capacity. The activities of lysozyme and SOD1 will be determined using standard assays with minor modifications. Site-directed spin labeling will then be utilized to implant spin probes into each model protein. EPR dynamics and long-range distance distribution measurements will then be carried out to determine global and local (the active site and/or substrate pathway) conformational flexibility changes of the two model proteins confined in different MOFs/COFs. SDSL-EPR is ideal for these experiments because it can overcome the complexities caused by MOF/COF backgrounds and protein-MOF/COF interactions and reveal the needed structural information. Correlating the function with the structural details under varied confined conditions will establish the correlation between confinement factors and protein structure-function relationship. This project is jointly funded by the Molecular Biophysics cluster of the Division of Molecular and Cellular Biosciences (MCB) and the Established Program to Stimulate Competitive Research (EPSCoR).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.