Structure. 2007 Dec; 15(12):1663-73.

Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

Protein Expression and Purification, Vol. 56, 110-120, 2007

Eukaryotic—especially human—membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.

FEMS Microbiology Letters, Volume 297, Issue 1, Date: August 2009, Pages: 87-94

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N2. Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N2 production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N2 and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.

Angew Chem Int Ed Engl. 2009;48(9):1656-9.

A simple and scalable method is presented for harvesting, purification, and on-chip processing of mammalian plasma membrane vesicles (PMVs) optimized for downstream proteome analysis. After immobilization on a microfluidic flowcell of PMVs, the embedded membrane proteins are proteolytically digested, and the peptides harvested and analyzed by LC-MS/MS. Over 93% of the detected proteins are plasma-membrane-derived.