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

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.