PROBING THE NATURE OF NONCOVALENT INTERACTIONS BY MASS-SPECTROMETRY -A STUDY OF PROTEIN-COA LIGAND-BINDING AND ASSEMBLY

A series of noncovalent complexes formed between the 86 residue acyl C oA binding protein (ACBP) and a series of acyl CoA derivatives has bee n studied by electrospray ionization mass spectrometry. Conditions wer e found under which CoA ligands can be observed in the mass spectromet er 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-t o-protein concentration in the electrospray solution were found to inc rease the proportion of intact complex observed in the spectrum. Varia tion in the length of the hydrophobic acyl chain of the ligand (C-16, C-12, C-8, C-0) resulted in similar proportions of complex observed in the mass spectrum even though significant variation in solution disso ciation constants has been measured. A substantially reduced proportio n 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 t he different factors stabilizing complexes in the gas phase environmen t of the mass spectrometer, The complexed species were also investigat ed by hydrogen-deuterium exchange methods combined with mass spectrome tric analysis and the results show that folding of ACBP occurs prior t o complex formation in solution, The results also show increased hydro gen exchange protection in the complex when compared with the free pro tein, Furthermore, even after dissociation of the complex, under these nonequilibrium gas phase exchange conditions, increased protection fr om hydrogen exchange in the complex is maintained.