Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

Abstract

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. C almodulin is a ubiquitous eukaryotic protein that regulates the activity of a wide variety of enzymes through its binding to them in the presence of calcium [1,2]. The interaction between calmodulin and a target protein is a key step in many calcium-regulated cellular functions [3,4]. Calmodulin has also been shown to be able to bind a wide range of smaller ligands, including polypeptides, in which the interaction takes place between amino acid side chains [5,6]. Peptides with little sequence homology, however, are capable of binding to calmodulin, though the majority of peptides that do so have a high propensity to form an amphipathic ␣-helix [7,8] and contain a cluster of basic residues.The interaction between calmodulin and the ␣-helical peptide melittin has been the subject of a number of investigations over the years using a range of spectroscopic approaches [9 -17]. Fluorescence and circular dichroism spectroscopy have revealed that in the presence of calcium, both calmodulin and melittin undergo significant structural changes, particularly in the vicinity of tyrosine residues 99 and 138 in calmodulin and at tryptophan 19 in melittin [9]. Small angle X-ray scattering has also identified significant structural changes in calmodulin with its transformation from a dumbbell to globular conformation upon its binding to melittin [13]. Similar conclusions have been drawn from limited proteolysis experiments followed by mass spectrometric analysis [16].Despite an earlier report on the successful growth of crystals of the calmodulin-melittin complex containing bound calcium [18], no X-ray crystallographic data have been made publicly available. X-ray crystal data for other calmodulin-peptide complexes, however, have been obtained [19] and the structure of the calmodulinmelittin complex predicted [16] based on this data and proton NMR studies for the unbound protein [20]. Nonetheless, to-date there exists no high-resolution experimental data for the calmodulin-melittin complex that represents an important model of calmodulin target recognition.To explore further the interaction between this im-

Oxidative protein labeling in mass-spectrometry-based proteomics

“…Alternatively, the electrospray emitter of a mass spectrometer’s source can be employed to oxidize atmospheric oxygen. High voltage on the electrospray emitter tip (typically above 5 kV) can generate a corona discharge, which leads to hydroxyl and perhydroxyl radical formation and subsequent peptide oxidation in the gas phase [79], an approach that has been used for protein footprinting experiments [80, 81] (see “Protein surface mapping”). However, Boys et al [82] showed recently that corona discharge can occur under regular electrospray ionization (ESI) conditions (3.5 kV, N 2 nebulizer gas) and induce hemoglobin oxidation.…”

Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

“…40,41 Most of the oxidative modifications generated in this way are easily recognizable as + 16-Da modifications in the resulting mass distributions, but less abundant products associated with other mass shifts may also be formed. [41][42][43][44] Oxidative labeling has been used for the structural mapping of numerous water-soluble biomolecular systems, 41,43,[45][46][47] as well as a few membrane proteins. 48,49 Our group recently applied ·OH labeling to native purple membranes.…”

Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

“…The first successful application of the oxidative footprinting to study of protein complexes was in the case of the calcium dependent interactions of actin-gelsolin 49 , and calmodulin-mellitin 50 . In this case the radicals were generated in situ by coupling electrical discharges with electrospray MS.…”