Designing an ATP-binding protein by harnessing His-tag in secondary structure

Abstract

Adenosine triphosphate (ATP) is an organic molecule that powers the cellular functions of all living organisms. Harnessing energy from ATP requires an enzyme to cleave off the third phosphate group. The detailed mechanisms of how an enzyme binds to the ATP molecule before this reaction should be better understood to provide the basis for advanced biological research. Since ATP has numerous complex intramolecular interactions, the adenine and the phosphates will be studied independently from each other. Although current literature contains some relevant information, such as the extensively studied Walker folds in ATP-binding proteins, this literature can be difficult to interpret due to the potential impact of evolution. Through investigation using De Novo protein designs, a new ATP-binding protein will be designed. The design of an adenine-binding protein will be studied first. Proteins are synthesized by inducing protein synthesis in living cells. After protein synthesis, a Ni-NTA column is used to isolate desired protein from other proteins that were also produced in synthesis. The his-tag residue on the desired protein and the imidazole in the elution buffer interact with the nickel resin to isolate the desired protein. Next, the protein is purified using the TEV protease to cleave protein at the TEV cleavage site and remove the his-tag. The Ni-NTA column is used once again to separate desired protein from cleaved parts. SDS-PAGE confirmed the effective synthesis and purification of proteins. After purification, we characterized the binding and protein structure. The use of circular dichroism (CD) allows further confirmation of the secondary structure of the purified proteins. Isothermal titration calorimetry (ITC) provides information regarding the entropy, enthalpy, and affinity of the adenine-binding. Titrations of adenine studied using ultraviolet–visible spectroscopy (UV-Vis) show that adenine will not aggregate, ensuring that the results of the ITC binding studies will not be disrupted by aggregation of adenine. Further UV-Vis titrations of adenine in protein have confirmed binding. Pursuit to develop an ATP-binding protein will provide crucial information regarding the phosphorylation process and enhance the understanding of physiology of biological organisms.