Structural characterization of protein-protein interaction sites by FT-ICR MS

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The interaction between cystatin and papain was investigated by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision induced dissociation (CID) in a hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain examining the mass of each fragment ion produced by hexapole-CID.In the absence of papain, amide hydrogens in short amino-terminal fragments were highly deuterated within 1min. In the case of cystatin-papain complex, significant drops in initial deuterium contents were recognized throughout the sequence of cystatin. Remarkable reduction in deuterium content in the region of residues l-10 was recognized for hours, suggesting that the flexible N-terminal region should have been tightly fixed in the binding pocket with hydrogen bonds. These resu … More lts were consistent with the previous studies on the structure and inhibition mechanism of cystatin. It was demonstrated that protein-protein interaction can be characterized by H/D exchange in combination with CID using ESI-FTICR MS within a short time using a small amount of sample.ESIMS was applied to the complex of papain and cystatin with partly lagged N-terminus. When cystatin was mixed with equimolar quantity of papain, the relative intensity of the free full-length cystatin dramatically decreased. It might be caused by the higher binding affinity of the intact cystatin for papain than those of the truncated forms. These results were consistent with those of the H/D exchange of cystatin-papain complex.The structural change of a peptide, melittin, caused by the interaction with lipid-micelles, dodecylphosphocholine, was investigated using the same method. It was confirmed that melittin is in a stable conformation in the presence of the lipid, while its structure is very flexible in the absence of the lipid. Less