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

Abstract

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.