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

Adenylate kinase (AK) from the thermoacidophilic archaeon Sulfolobus acidoc aldarius was cloned and overexpressed in E. coli to allow large scale prepa rations for detailed biophysical studies. Oligomerization of its subunits a nd its substrate binding specificity to adenosine nucleotides were investig ated by matrix-assisted laser desorption/ionization mass spectrometry (MALD I-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 p reparation from Sulfolobus acidocaldarius was found by a specific 'first sh ot' MALDI-MS strategy to be a homotrimer. The non-covalent interactions bet ween 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 differen t extents. For observations pf specific interactions between proteins and t heir ligands of much lower molecular mass by MALDI-MS, the choice of the pr oper matrix and the accumulation of first shot spectra separately from foll owing shot spectra are crucial to obtain data which might reflect vital int eractions in living cells.