Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry.
The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.
Wong
Jason W H
JW
School of Molecular and Microbial Biosciences, The University of Sydney, Sydney, Australia.
Maleknia
Simin D
SD
Downard
Kevin M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000378:metabolism; D008555:Melitten / Q000737:chemistry* / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D015335:Molecular Probes; D016877:Oxidants; D008555:Melitten; D017665:Hydroxyl Radical; D014364:Tryptophan