Investigating the role of allosteric regulation in ATP homeostasis
Denis Titov, Assistant Adjunct Professor
Nutritional Sciences and Toxicology
Applications for Fall 2024 are closed for this project.
ATP powers most energy-consuming reactions in cells. While ATP has a short half-life, the demand for ATP also fluctuates. Thus, ATP or energy homeostasis in cells needs to be appropriately regulated. ATP homeostasis has to perform at least three tasks: allow energy generation from ATP hydrolysis, maintain stable and high ATP concentration, and match ATP production rate with cellular demand. This project focuses on ATP production through glycolysis and the rapid allosteric regulation controlling this pathway. Four enzymes in glycolysis are allosterically regulated and postulated to play a critical role in regulating the rate of glycolytic ATP production. However, this postulate has never been experimentally tested, and it remains unclear whether allosteric regulation is required to control the rate of ATP production or any other of the fundamental properties of ATP homeostasis. To investigate the problem, our lab has developed a mathematical model that simulates glycolysis activity. The result from our model, contrary to the textbook view that allostery regulates the rate of ATP production, predicted that allosteric regulation is not required to match ATP production and consumption and allow energy generation from ATP hydrolysis, while maintenance of high and stable ATP levels required allosteric regulation on HK and PFK. The aim of this project is to use in vitro reconstituted glycolysis, human cell lines, and mutant enzymes devoid of allosteric regulation to test the prediction that allosteric regulation is required to regulate ATP levels but not the ATP production rate or energy of ATPase reaction.
Role: The main role of the undergraduate student will be to prepare material and perform reconstituted in vitro assay, though the exact responsibilities are negotiable. The student will be exposed to techniques such as protein preparation through affinity or IEX chromatography, western blotting, and in vitro enzyme activity assay. The student will also learn how to properly process, and analyze data generated.
If interest and productivity persist throughout the semester, there will be opportunities to continue this project in future semesters. With greater project involvement, independent research can as the foundation for an honors thesis. All contributions to the advancement of the project will lead to inclusion as an author in future presentations and publications.
Qualifications: 1) highly motivated and enthusiastic in the field of metabolism, 2) organized in time, 3) perseverant and hardworking, 4) open to ask questions and communicate clearly.
Previous experience in research is not required, although experience in biochemistry or molecular biology lab will be considered valuable. This project has experiments that last for multiple consecutive days, therefore, the amount of time available and being organized will be taken into consideration.
Day-to-day supervisor for this project: Xinyi Yang, Ph.D. candidate
Hours: 12 or more hours
Related website: http://denistitovlab.org
Biological & Health Sciences