Examining the Binding Affinity of Ribose to YME1L-AAA+ By Competition Titrations

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West, Justin
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University Honors College Middle Tennessee State University
YME1L is an ATP-dependent protease that is involved in both protein quality control and regulation of mitochondrial morphology. One method to detect binding interactions of this protein is through the use of a fluorescently tagged ATP, known as MANT-ATP. ATP is made of a sugar, ribose, a triphosphate group, and a nitrogenous base, adenine. Binding assays that utilize MANT-ATP have been widely used, but sometimes questioned due to possible non-specific binding interactions. The purpose of this study is to look at the difference in interactions between unmodified ATP and the fluorescent analog MANT-ATP. Results show that MANT-ATP binds to not only the active site of the protein, but possibly multiple sites due to non-specific interactions occurring at high MANT-ATP concentration. Furthermore, we look at how ATP binding is affected by the presence of ribose to look at a possible competition effect. These results show that ribose is in fact able to bind to the ATP binding site alone without the presence of adenine or the phosphates.
College of Basic and Applied Sciences, Mitochondria, YME1L, AAA+, ATP, Ribose, MANT-ATP, titration, equilibrium constant, competition, specific interaction, FRET, tryptophan, fluorescence