Probing the nature of noncovalent interactions by mass spectrometry. A study of protein-CoA ligand binding and assembly
Robinson CV., Chung EW., Kragelund BB., Knudsen J., Aplin RT., Poulsen FM., Dobson CM.
A series of noncovalent complexes formed between the 86 residue acyl CoA binding protein (ACBP) and a series of acyl CoA derivatives has been studied by electrospray ionization mass spectrometry. Conditions were found under which CoA ligands can be observed in the mass spectrometer bound to ACBP. Despite the very low dissociation constants (10-7 to 10-10 M) of the acyl CoA ligand complexes high ratios of ligand-to-protein concentration in the electrospray solution were found to increase the proportion of intact complex observed in the spectrum. Variation in the length of the hydrophobic acyl chain of the ligand (C16, C12, C8, C0) resulted in similar proportions of complex observed in the mass spectrum even though significant variation in solution dissociation constants has been measured. A substantially reduced proportion of complex was, however, found for the mutant proteins, Y28N, Y31N, and Y73F, lacking tyrosine residues involved in critical interactions with the CoA ligand. These results have been interpreted in terms of the different factors stabilizing complexes in the gas phase environment of the mass spectrometer. The complexed species were also investigated by hydrogen-deuterium exchange methods combined with mass spectrometric analysis and the results show that folding of ACBP occurs prior to complex formation in solution. The results also show increased hydrogen exchange protection in the complex when compared with the free protein. Furthermore, even after dissociation of the complex, under these nonequilibrium gas phase exchange conditions, increased protection from hydrogen exchange in the complex is maintained.