We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.Keywords: Escherichia coli; membrane protein overexpression; membrane protein isolation; membrane protein characterization; GFP Membrane proteins (MPs) account for 20%-25% of all open reading frames in sequenced genomes, and fulfill a wide range of central functions in the cell (Wallin and von Heijne 1998). However, our knowledge of this important class of proteins is still poor, mainly because of a lack of generally applicable approaches to the overexpression and purification steps that precede functional and structural analysis. Novel approaches in these areas are required to facilitate and speed up MP research.The bacterium Escherichia coli is still the most widely used vehicle for MP overexpression. Overexpression in the cytoplasmic membrane is preferred to overexpression in inclusion bodies, since the isolation of functional MPs from the membrane is usually more successful than refolding from inclusion bodies (Drew et al. 2003). Green fluorescent protein (GFP) fusions can be used to facilitate the monitoring of MP overexpression in the cytoplasmic membrane (Drew et al. 2001). If the fusion protein ends up in inclusion bodies, GFP does not fold and is therefore not fluorescent; in contrast, if the fusion is expressed in the cytoplasmic membrane, GFP folds properly and is fluorescent. GFP is only fluorescent in the cytoplasm of Escherichia coli (Drew et al. 2002), which means that GFP-based screens work only for MPs that have their C terminus located in the cytoplasm. Recently, nearly all E. coli cytoplasmic MPs were fused to GFP for a membrane proteome topology screen (Daley et al. 2005). Approximately 80% of all E. coli cytoplasmic MPs have a cytoplasmic C terminus, and thus GFP can be used to monitor the overexpression levels of the majority of E. coli MPs (Daley et al. 2005).Here, we present a generic pipeline for rapid overexpression screening, detergent extraction, and purification of MPs based on a simple MP-GFP fusion approach. We show that milligram amounts of pure functional MP can Reprint requests to: Jan-Willem de Gier, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; e-mail: degier@dbb.su.se; fax: +46-8-153679.Article published online ahead of print. Article and publication date are at

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The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47°C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.Keywords: chemical cross-linking; mass spectrometric peptide mapping; small heat-shock protein; protein-protein interactions; citrate synthase Small heat-shock proteins (sHsps) exist in essentially all organisms and are especially abundant in plants (Waters et al. 1996;Haslbeck et al. 2005). The sHsps have a particularly important role during heat stress, where they protect other proteins from aggregation (Horwitz 1992; Narberhaus 2002) and prevent irreversible accumulation of large aggregates within the cell (Sun and MacRae 2005). By maintaining unfolded proteins in a folding-competent state, sHsps work with the ATP-dependent Hsp70 systems, which can then refold the proteins when the stress conditions return to normal (van Montfort et al. 2001b;Haslbeck 2002;Stromer et al. 2003;Haslbeck et al. 2004).The sHsp monomers range from 12 to 43 kDa and assemble into large multimers (van Montfort et al. 2001b;Haslbeck et al. 2005). The three-dimensional structures have been determined for the plant Hsp16.9 from wheat Reprint requests to: Emma Å hrman, Department of Biochemistry, Lund University, P.O.Box 124, S-22100, Lund, Sweden; e-mail: emma. ahrman@biochemistry.lu.se; fax: 46-46-222-4534.Abbreviations: DTSSP, 3,39-Dithiobis(sulfosuccinimidylpropionate); DTT, dithiothreitol; MDH, malate dehydrogenase; CS, citrate synthase; nanoESI-MS, nanoelectrospray-ionization mass spectrometry.Article published online ahead of print. Article and publication date are at

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25°in relation to each other, suggesting a role for global dynamics in dodecamer function.

Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.