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 homodimer.glutathione 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.
Highlights
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
Secondary structure elements and structural dynamics can be studied by mass spectrometry in hydrogen/deuterium exchange experiments, in which the exchange kinetics of amide protons are measured [21,22,23], making ES mass spectrometry an attractive tool for studies of protein structure and organization
This work showed the feasibility of directly measuring hydrogen/deuterium exchange kinetics of detergent-solubilized proteins in solution by ES mass spectrometry and thereby obtaining structural information
Summary
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. ES has been extensively used in the direct study of noncovalent interactions, the application of matrix-assisted laser desorption ionization (MALDI) in this area of research been more restricted, it should be noted that Van Dorsselaer et al [33, 34] have reported MALDI data on the subunit stoichiometry of high mass noncovalent complexes of membrane proteins. The ES interface conditions were optimized for the preservation of noncovalent protein complexes while allowing desolvation from the MGST1 protein complex. “in-source” collision-induced dissociation of the complex was performed This allowed the noncovalent binding of GSH to the complex to be investigated. To the best of our knowledge, the results presented here represent the first application of ES mass spectrometry to the direct analysis of a biologically important noncovalently bound membrane protein complex
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