Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.

Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination.

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a ß sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization.

Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5×102 to 1.8×104 m-1 s-1 . Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.

Large conformational changes of a highly dynamic pre-protein binding domain in SecA.

SecA is an essential molecular motor for the translocation of proteins across the membrane via the bacterial Sec secretion system. While the Sec system is found in all cells from archaea to multicellular eukaryotes, the SecA protein is mainly found in bacteria. The mechanism of how the motor protein works on a molecular level is still under dispute but it is well established that SecA binds ATP and uses its hydrolysis for the translocation of substrates. In this work, we addressed the question of which conformational changes the protein might undergo during protein translocation. To this end, we investigated the molecular movements of SecA in the absence or the presence of ATP using single-molecule FRET measurements and in silico normal mode analyses. Our results demonstrate that the pre-protein binding domain of SecA is highly dynamic in the absence of the nucleotide and moves towards the helical wing domain in an ATP-bound state.

Central alveolar hypoventilation (Ondine's curse) caused by megadolichobasilar artery.

Central alveolar hypoventilation (CAH) syndrome is a clinical condition that is characterized by the loss of automatic breathing, particularly during sleep. Most forms in adults are caused by brainstem ischemia, mass, infection, demyelinating disease, or anoxic-ischemic damage. We present a case of a fatal symptomatic acquired CAH syndrome caused by megadolichobasilar artery. A 62-year-old man with pre-existing vascular dementia suffered an acute posterior stroke. During stroke care, long episodes of hypopnea and apnea were observed which responded well to verbal reminders. During an unobserved episode, the patient was found unresponsive, with chemical signs of prolonged hypoventilation not explainable by cardiopulmonary disease. A diaphragmatic pacemaker, assisted ventilation, and repeated resuscitation were refused by the patient, who died some days later. CAH is a rare complication that can occur in patients with megadolichobasilar artery.

Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models.

Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present.

Cerebral air embolism via port catheter and endoscopic retrograde cholangio-pancreatography.

BACKGROUND: Cerebral air embolism (CAE) is a critical clinical condition necessitating rapid diagnosis and therapeutic measures. METHODS: The authors describe two patients with lethal CAE. RESULTS: An 81-year-old man rapidly developed coma with tetraplegia. CT-scan revealed prominent CAE whereby the entry of the air was via a port catheter for parenteral nutrition. A 45-year-old man with severe alcohol-toxic multiple organ damage needed endoscopic retrograde cholangio-pancreatography (ERCP) and a second esophagogastroscopy. After an epileptic seizure, the CT-scan of the brain showed small amounts of cerebral air in the posterior right hemisphere and in the sagittal superior sinus. Despite critical care the patient died. CONCLUSION: CAE is a neurocritical emergency case. Early CT-scan of the brain can detect air, guide further therapy, and contribute to the assessment of the prognosis.

Real time observation of single membrane protein insertion events by the Escherichia coli insertase YidC.

Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.

Efficient conversion of arylene precursors into photoluminescent phosphonates for surface modification of metal oxides.

Three highly fluorescent phosphonates have been prepared in good yields from different arylene bridged 5-iodothiophenes by following an optimized four-step procedure. The compounds have been immobilised on mesoporous zirconia, alumina and titania particles in order to probe their luminescence properties on the surfaces. The organic compounds as well as the obtained hybrid materials have been characterised using liquid phase or MAS NMR spectroscopy, N2 physisorption measurements, ATR-IR spectroscopy and elemental analysis. The luminescence intensity of the organic molecules was found to be in general much more dependent on the surface used for grafting rather than on the dye loading obtained for different metal oxides powders. The luminescence extinction, which has been detected for all coated TiO2 samples, is most likely due to the different electronic properties of this semiconducting support compared to the other surfaces. The method described shows how fluorescent tracers can be easily synthesised and applied in surface analytics after exploring the interaction of the corresponding organophosphonates with several ceramic interfaces.

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer.

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Three-color Förster resonance energy transfer within single F0F1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.

Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary F(O) motor connected to a 3-stepped F(1) motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

Dynamic ligand-induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy.

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10-100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.

Novel acridone-modified MCM-41 type silica: Synthesis, characterization and fluorescence tuning.

A Mobil Composition of Matter (MCM)-41 type mesoporous silica material containing N-propylacridone groups has been successfully prepared by co-condensation of an appropriate organic precursor with tetraethyl orthosilicate (TEOS) under alkaline sol-gel conditions. The resulting material was fully characterized by means of X-ray diffraction (XRD), N(2)-adsorption-desorption, transmission electron microscopy (TEM), IR and UV-vis spectroscopy, as well as (29)Si and (13)C CP-MAS NMR techniques. The material features a high inner surface area and a highly ordered two-dimensional hexagonal pore structure. The fluorescence properties of the organic chromophore can be tuned via complexation of its carbonyl group with scandium triflate, which makes the material a good candidate for solid state sensors and optics. The successful synthesis of highly ordered MCM materials through co-condensation was found to be dependent on the chemical interaction of the different precursors.

YidC-driven membrane insertion of single fluorescent Pf3 coat proteins.

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.

Recurrent tonsillitis in adults: quality of life after tonsillectomy.

BACKGROUND: The aim of this study was to assess the effect of tonsillectomy in adults with recurrent tonsillitis on their quality of life and on their use of medical resources. METHOD: 114 patients who had had at least three episodes of acute tonsillitis in the 12 months preceding tonsillectomy were evaluated pre- and postoperatively with a questionnaire developed by the authors, and with the Glasgow Benefit Inventory. RESULTS: 97 patients (85%) filled out the questionnaires completely. The Glasgow Benefit Inventory revealed an improvement in the overall score (+19) and in the partial scores for general well-being (+18) and physical health (+39). The degree of support from friends and family was unchanged (±0). Significant decreases were observed in visits to a physician, analgesic and antibiotic consumption, days off from work, and episodes of sore throat. The number of visits to a physician because of sore throat decreased from an average of five preoperatively to one postoperatively; the number of episodes of sore throat, from seven to two; and the number of days taken off from work, from twelve to one per year. 65% of the patients surveyed took analgesics for sore throat preoperatively, 7% postoperatively. 95% took antibiotics for sore throat preoperatively, 22% postoperatively. CONCLUSION: Although this study had a number of limitations (small size, retrospective design, short follow-up), it was able to show that tonsillectomy for adults with recurrent tonsillitis improves health and quality of life and reduces the need to consume medical resources.