Baculoviral expression of an integral membrane protein for structural studies.

The baculovirus system has proven successful for the expression of integral membrane proteins for structural studies. A recombinant baculovirus, in which the gene of interest is placed under the control of the late-stage polyhedrin promoter, serves as the starting point for viral expansion and protein expression studies. Using large-scale insect cell culture techniques together with a filter-binding assay for protein function, the conditions of expression, purification, and solubilization can be optimized. As applied to the glutamate receptor ion channel subunit GluR2, this approach yields milligram quantities of pure, active protein, which have been used for single-particle electron microscopic analysis of the receptor structure. Detergent exchange protocols are also discussed, as a prerequisite for two-dimensional crystallization trials.

The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly.

The ionotropic glutamate receptors (iGluRs) represent a major family of ion channels whose quaternary structure has not yet been defined. Here, we present the three-dimensional structure of a fully assembled iGluR, determined at approximately 20A resolution by electron microscopy. Analysis of negatively stained single-particle images reveals the presence of 2-fold, but not 4-fold, symmetry for these tetrameric channels, providing the first direct structural evidence for a dimer-of-dimers assembly. The receptor appears elongated, measuring approximately 170Ax140Ax110A, with the 2-fold symmetry centered on its longitudinal axis. The overall molecular shape and symmetry suggest an orientation relative to the membrane and permit the identification of a putative transmembrane domain. Internal cavities located along the longitudinal axis may represent components of the ion conduction pathway.

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution.

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.

Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies.

Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.