Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors.

G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.

Linear and extended: a common polyglutamine conformation recognized by the three antibodies MW1, 1C2 and 3B5H10.

A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies-MW1, 1C2 and 3B5H10-have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

Thioether analogues of disulfide-bridged cyclic peptides targeting death receptor 5: conformational analysis, dimerisation and consequences for receptor activation.

Cyclic peptides containing redox-stable thioether bridges might provide a useful alternative to disulfide-bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16-mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL-R2). Upon covalent oligomerisation, the disulfide-bridged peptide has previously shown similar behaviour to that of TNF-related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5-binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5-mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.

Surface plasmon resonance analysis of the binding mechanism of pharmacological and peptidic inhibitors to human somatic angiotensin I-converting enzyme.

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two K(D)'s differing by a factor 10-20 and >30, respectively (lowest K(D) = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with K(D) in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC50 of 4.3 µM, bound to ACE with K(D) = 3-4 µM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.

Huntingtin affinity for partners is not changed by polyglutamine length: aggregation itself triggers aberrant interactions.

Huntington's disease (HD) is caused by the expansion mutation above a length threshold of a polyglutamine (polyQ) stretch in the huntingtin (Htt) protein. Mutant Htt (mHtt) pathogenicity is proposed to rely on its malfunction and propensity to misfold and aggregate. Htt has scaffolding properties and has been reported to interact with hundreds of partners. Many interactors show apparent increased or decreased affinity (dysinteraction) for mHtt, which may account for selective malfunctions and striatal degeneration in HD. These dysinteractions are proposed to result from mutant polyQ conformational changes that remain elusive. To date, dysinteractions have only been studied using semi-quantitative techniques with their outcome potentially influenced by the presence of mHtt aggregates. Therefore, the molecular mechanism underlying these dysinteractions remains to be determined. Here, we have used purified proteins devoid of aggregates to quantify the interaction of normal and mHtt with two partners: SH3GL3, reported to have increased binding to mHtt, and the 2B4 antibody, a model partner. Using surface plasmon resonance and pull-down techniques, we show that in the absence of aggregation polyQ length has no effect on Htt interactions. We demonstrate that the presence of aggregates affects the spatial distribution and solubility of Htt partners and strongly influences the outcome of pull-down experiments. Our results show that expanded polyQ per se does not alter Htt interactions and suggest that aggregated mHtt form molecular platforms that influence the Htt interacting network. Modulating mHtt aggregation could thus have beneficial effects on specific cellular pathways deregulated in HD.

Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.

We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 µM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 µM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 µM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 µM range. We propose that a medium (µM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

Validation of surface plasmon resonance screening of a diverse chemical library for the discovery of protein tyrosine phosphatase 1b binders.

We investigated the suitability of surface plasmon resonance (SPR) for providing quantitative binding information from direct screening of a chemical library on protein tyrosine phosphatase 1b (PTP1B). The experimental design was established from simulations to detect binding with K(D) < 10⁻4 M. The 1120 compounds (cpds) were injected sequentially at concentrations [C(cpd)] of 0.5 or 10 µM over various target surfaces. An optimized evaluation procedure was applied. More than 90% of cpds showed no detectable signal in four screens. The 30 highest responders at C(cpd)=10 µM, of which 25 were selected in at least one of three screens at C(cpd)=0.5 µM, contained 22 promiscuous binders and 8 potential PTP1B-specific binders with K(D) ~10⁻5 M. Inhibition of PTP1B activity was assayed and confirmed for 6 of these, including sanguinarine, a known PTP1B inhibitor. C(cpd) dependence studies fully confirmed screening conclusions. The quantitative consistency of SPR data led us to propose a structure-activity relationship (SAR) model for developing selective PTP1B inhibitors based on the ranking of 10 arylbutylpiperidine analogs.

GPRASP/ARMCX Protein Family: Potential Involvement in Health and Diseases Revealed by their Novel Interacting Partners.

GPRASP (GPCR-associated sorting protein)/ARMCX (ARMadillo repeat-Containing proteins on the X chromosome) family is composed of 10 proteins, whose genes are located on a small locus of the X chromosome except one. They possess at least two armadillo-like repeats on their carboxylterminal homologous sequence, but they can be subdivided on specific sequence features. Subfamily 1 (GPRASP1, GPRASP2, GPRASP3, ARMCX4 and ARMCX5) displays additional repeated motifs while a mitochondrial targeting transmembrane domain is present in subfamily 2 (ARMC10, ARMCX1, ARMCX2, ARMCX3 and ARMCX6). Although their roles are not yet fully understood, the recent identification of several interacting partners has shed new light on the processes in which GPRASP/ARMCX proteins are implicated. Among the interacting partners of proteins from subfamily 1, many are GPCRs. GPRASP1 binds trafficking proteins, such as Beclin2 and the Dysbindin-HRS-Gαs complex, to participate in GPCR post-endocytic sorting. Moreover, in vitro as well as in vivo experiments indicate that GPRASP1 is a critical player in the adaptive responses related to chronic treatments with GPCR agonists. GPRASP2 seems to play a key role in the signaling of the hedgehog pathway in the primary cilium through a Smoothened-GPRASP2-Pifo complex. Identified small compound inhibitors of this complex could treat drug-resistant smoothened derived cancer forms. Deletion of GPRASP2 in mice causes neurodevelopmental alteration and affects mGluR5 regulation, reflected by autism-like behavior. Several members of subfamily 2, in complex with TRAK2 and MIRO, are involved in the trafficking of mitochondria in axons and in the regulation of their size and division, influencing the cell cycle. The essential role of GPRASP/ARMCX proteins in cellular physiology is supported by human cases of deletions, causing male neonatal lethality by pulmonary delayed development, dysmorphic face, and psychiatric and intellectual impacts in females.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Arthritogenic monoclonal antibodies from K/BxN mice.

Arthritis in the K/BxN mouse model is provoked by pathogenic antibodies (Abs) directed against a ubiquitously expressed protein, glucose-6-phosphate isomerase (GPI). To begin dissecting the repertoire of arthritogenic immunoglobulins (Igs) in the K/BxN model, and to provide a basis for comparison with RA patients we have generated anti-GPI monoclonal Abs (mAbs) from spontaneously activated B cells in the lymphoid organs of arthritic mice. B cell clones with anti-GPI specificities were present at extraordinarily high frequencies in the spleen, and less frequently in other lymphoid organs and in the synovial fluid. None of the anti-GPI mAbs induced arthritis when injected individually into healthy recipients, but most were effective when combined in pairs or larger pools. Arthritogenic combinations depended on mAbs of the IgG1 isotype, which bound to GPI with Kd in the 10(-9) M range, with no indication of cooperative binding between complementing pairs. Pathogenicity was not associated with recognition of a particular epitope, but the ability to form mAb/GPI multimers by simultaneous recognition of different epitopes was clearly required, consistent with the known role of complement and FcRs in this model. Sequence analysis revealed structural similarities amongst the mAbs, indicating that a particular subset of B cells may evade tolerance in K/BxN mice, and that affinity maturation by somatic mutation likely takes place. These results confirm that GPI itself, rather than a cross-reactive molecule, is the target of pathogenic Igs.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates.

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA.

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.

Pathogenic and non-pathogenic polyglutamine tracts have similar structural properties: towards a length-dependent toxicity gradient.

Abnormally expanded polyglutamine (polyQ) tracts provide a gain of toxic functions to nine otherwise unrelated human proteins and induce progressive neurodegenerative diseases. Over the past ten years, it was suggested that only polyQ tracts longer than a specific threshold adopt a particular structure, which would be the cause of the apparent polyQ length-dependent toxicity threshold observed in polyQ diseases. We have used a combination of biochemical and biophysical approaches to compare the structural properties of polyQ of pathogenic and non-pathogenic lengths under various conditions. We observe that pathogenic and non-pathogenic polyQ, as soluble species and upon interaction with a partner, during aggregation, or as mature aggregates, display very similar structural properties. PolyQ length only influences the aggregation kinetics and, to a lesser extent, the stability of the aggregates. We thus propose that polyQ toxicity does not depend on a structural transition occurring above a specific threshold, but rather that polyQ tracts are inherently toxic sequences, whose deleterious effect gradually increases with their length. We discuss how polyQ properties and other cellular factors may explain the existence of an apparent polyQ length-dependent toxicity threshold.

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity.

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.

Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen.

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells.

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.