Identifying nucleoside and nucleotide protein interactions with S-adenosyl-L-homocysteine and adenosine triphosphate capture compounds

The current work aims to contribute to the proteome research, which means the ultimate characterization of all proteins that are encoded by the genome and expressed in the cell. The work described in this thesis utilizes a method of functional proteomics, called Capture Compound Mass Spectrometry™. In this method, small synthetic trifunctional molecules are used as chemical probes for functional isolation and identification of certain classes of enzymes. A capture compound (CC) has a) a selectivity function (e.g., enzyme substrate or inhibitor) for specific targeting the active site of an enzyme, b) a reactivity function (a photo-crosslinker) for covalent attachment to the protein under UV light, and c) a sorting function (e.g., biotin) for isolation of the captured protein. This makes it possible to analyze these proteins with SDS-PAGE or identify them via LC-MS/MS. In the present work, CCs were designed and/or applied to target two important enzyme families: methyltransferases (MTases) and ATP-binding proteins (ABPs). The CC for MTase isolation had S-Adenosyl-L-homocysteine as selectivity function, a phenyl azide as reactivity function and a terminal alkyne as a chemical handle for the sorting function. The latter could be attached to the CC after the UV photo-crosslinking via Click chemistry. As a Click reaction, the copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition was used. The Click-SAH CC was successfully tested for its ability to capture the MTase MTaq.I. Various conditions of the capture process, such as detection method (fluorescence or streptavidin beads isolation), the length of the “sorting linker”, as well as the cycloaddition conditions, were optimized. In the second project, a series of gamma-ATP CCs for isolation of nucleoside kinases and other ABPs were tested. The CCs differed in terms of reactivity functions (phenyl azide, tetrafluorinated phenyl azide and trifluoromethyl diazirine) and the structure of the linker between the ATP and the rest of the molecule. The gamma-ATP CCs showed very different profiles towards the purified nucleoside kinases and towards the ABPs from E. coli lysate. From E. coli lysate, 62 ABPs were specifically isolated, among them known ATP-binders, but also as four uncharacterized proteins, thymidylate kinase (also captured in purified form) and Universal stress protein D, which was not known to be ATP-binding before