Oligomerization and substrate binding studies of the adenylate kinase from Sulfolobus acidocaldarius by matrix-assisted laser desorption/ionization mass spectrometry

Abstract

Adenylate kinase (AK) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was cloned and overexpressed in E. coli to allow large scale preparations for detailed biophysical studies. Oligomerization of its subunits and its substrate binding specificity to adenosine nucleotides were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The ‘first shot phenomenon’ in MALDI-MS, applied previously for the analysis of protein–protein interactions, was applied to study both the AK oligomerization and nucleotide binding. The native form of the AK protein preparation from Sulfolobus acidocaldarius was found by a specific ‘first shot’ MALDI-MS strategy to be a homotrimer. The non-covalent interactions between the enzyme and its substrates were also analysed by MALDI-MS. AK as a phosphotransferase binds to the three ligands, AMP, ADP and ATP, to different extents. For observations of specific interactions between proteins and their ligands of much lower molecular mass by MALDI-MS, the choice of the proper matrix and the accumulation of first shot spectra separately from following shot spectra are crucial to obtain data which might reflect vital interactions in living cells.