Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

Abstract

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer⅐glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys 49 (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144 -15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.Membrane proteins are estimated to constitute about onethird of the total proteome (1) and have important and diverse roles in biology, including functions as cell-surface receptors, ion channels, and transporters and in cell adhesion. However, because of the difficulty in producing crystals of detergentsolubilized proteins for x-ray crystallography, only ϳ1% of the unique protein structures so far solved at high resolution are of membrane proteins (2). Integral membrane proteins thus pose an important challenge in structural biology.Microsomal glutathione transferase-1 (MGST1) 1 is a member of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily. At present, other mammalian members of the MAPEG family are two glutathione transferases/peroxidases (MGST2 and MGST3), 5-lipoxygenase-activating protein, leukotriene C 4 synthase, and microsomal prostaglandin E 2 synthase (the closest relative to MGST1) (3). MGST1 functions both as a glutathione transferase and as a peroxidase, thus potentially protecting cells from reactive compounds and oxidative stress (3, 4). The quaternary structure of MGST1 has previously been investigated by electron crystallography (5, 6), radiation inactivation (7), cross-linking (8), and hydrodynamic (9) studies and has been suggested to be a homotrimer both in the microsomal membrane and in the purified form. Electrospray (ES) mass spectrometry has shown that the enzyme is partly N-acetylated (10). Interestingly, equilibrium dialysis (11) and stopped-flow active-site titrations (12) have indicated that the MGST1 homotrimer binds only one molecule of ...

MacB ABC Transporter Is a Dimer Whose ATPase Activity and Macrolide-binding Capacity Are Regulated by the Membrane Fusion Protein MacA

“…The development of strategies to tackle this field is challenging, primarily because the large quantities of detergent suppress the protein signal, whereas the poor solubility of membrane proteins in aqueous buffers often causes the electrospray needle to block. To date only a few MS studies have reported the observation of membrane proteins or their complexes by MS (35)(36)(37)(38)(39). Here we report on the use of a miniaturized form of ES with reduced flow rates (nano-ES), and a high collision energy that facilitates the desolvation process and induces dissociation of detergentproteins clusters, to determine the oligomeric state of an integral homomeric membrane protein.…”

Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione

“…Earlier we were able to perform a mass spectrometric analysis of intact MGST1 confirming the oligomeric structure and establishing that this technique was applicable to a membrane protein [23]. Technological improvements in the form of an automated nanoelectrospray method capable of high spray stability in the analysis of non-covalent complexes has now allowed us to study MGST1 ligand interactions by this technique demonstrating three bound GSH molecules per trimer.…”

Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry

“…While a vacuum can be regarded as hydrophobic and therefore a suitable environment in which to study such proteins [75], transferring them intact into the mass spectrometer has been a challenge. The first approach which brought success was to prepare a protein in a concentration of a detergent sufficient to solubilize the exposed hydrophobic surfaces, but not so high as to obscure the signal corresponding to protein [76]. …”