| 1. |
- Gorecki, Kamil
(författare)
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Antiporter-like protein subunits of respiratory chain complex I
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Complex I is the mitochondrial enzyme complex that oxidizes NADH produced in the citric acid cycle and reduces quinone in the membrane, coupled to proton pumping out of the mitochondrial matrix, creating a membrane potential. This process generates about 40% of the energy used by living organisms. High-resolution structures of complex I recently became available, but the molecular mechanism behind the coupling process remains unknown. This thesis is focused on better understanding of the three large membrane-spanning subunits that show primary sequence similarity to two proteins in Mrp-antiporters. The similarity suggests that these complex I subunits are somehow involved in transporting protons and/or other ions across the membrane, an essential element in the proton pumping/coupling functionality. First, a method of producing and purifying the NuoL, M, N and MrpA, D polypeptides in high amounts was developed. The C-terminal part of MrpA, that extends beyond the universally conserved 14 TM helices in the protein family, was further analysed and the transmembrane topology was determined. It was demonstrated that this domain in MrpA corresponds to the membrane parallel, so called lmp-helix in NuoL followed by the NuoJ subunit in complex I. Then, the purified antiporter-like polypeptides were reconstituted in liposomes, together with a novel pH sensitive probe. Glu3 is a dendritic porphyrin, which we have demonstrated can be enclosed inside lipid vesicles and used to accurately measure proton transport across membranes. Both the dye incorporation and protein insertion was successful, but the method requires further optimisation since the proteoliposomes were not tight enough to support build-up of a proton gradient. Subsequently, sodium interaction by the antiporter-like subunits was studied using Nuclear Magnetic Resonance (NMR), to investigate the putative interaction between sodium ions and the detergent-solubilised, purified protein molecules. No sodium binding was observed either for complex I subunits, nor for the control Mrp antiporter proteins. Then, the sodium translocation abilities were assessed in vivo in a Bacillus subtilis model system. The intracellular sodium content in a MrpA deletion strain, that exhibit a pH and sodium sensitive growth phenotype, was measured using 23Na-NMR and a membrane-impermeable shift agent. It was demonstrated that the growth defect was indeed caused by an inability to excrete sodium. Expression of Mrp proteins or homologous complex I subunits in the deletion strain was shown to influence the sodium homeostasis. A sequence motif in TM helix 8 that is present in MrpA/NuoL, but not in MrpD/NuoMN, that could possibly be responsible for the ion specificity of the respective subunits, was identified. The site-directed mutagenesis and subsequent functional analyses work of the motif is still ongoing, but some preliminary results are presented.
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| 2. |
- Górecki, Kamil, et al.
(författare)
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Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:34, s. 11831-11837
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of protein-facilitated copper flux across biological membranes is a considerable challenge. Here, we demonstrate a straightforward microfluidic-derived approach for visualization and measurement of membranous Cu flux. Giant unilamellar vesicles, reconstituted with the membrane protein of interest, are prepared, surface-immobilized, and assessed using a novel quencher-sensor reporter system for detection of copper. With the aid of a syringe pump, the external buffer is exchanged, enabling consistent and precise exchange of solutes, without causing vesicle rupture or uneven local metal concentrations brought about by rapid mixing. This approach bypasses common issues encountered when studying heavy metal-ion flux, thereby providing a new platform for in vitro studies of metal homeostasis aspects that are critical for all cells, health, and disease.
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| 3. |
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| 4. |
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| 5. |
- Gorecki, Kamil, et al.
(författare)
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The Na(+) transport in Gram-positive bacteria defect in the Mrp antiporter complex measured with (23)Na-NMR.
- 2014
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 445:Online 15 October 2013, s. 80-86
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Tidskriftsartikel (refereegranskat)abstract
- (23)Na-NMR has previously been used to monitor Na(+) translocation across membranes in Gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na-NMR method was adapted for measurements of internal Na(+) concentration in the Gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp antiporter complex, a member of the Cation Proton Antiporter-3 (CPA-3) family. The sodium sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted, could indeed be correlated to the inability of this strain to maintain a lower internal than external Na(+) concentration.
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| 6. |
- Gustavsson, Tobias, et al.
(författare)
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A cytochrome c- fusion protein domain for convenient detection, quantification and enhanced production of membrane proteins in Escherichia coli - expression and characterization of cytochrome-tagged complex I subunits
- 2010
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 19:8, s. 1445-1460
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Tidskriftsartikel (refereegranskat)abstract
- Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work the C-terminal ends of the complex I subunits NuoH, NuoL, NuoM and NuoN from E. coli complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared to other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.
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| 7. |
- Hasan, Kamrul, et al.
(författare)
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Electrochemical Communication Between Electrodes and Rhodobacter capsulatus Grown in Different Metabolic Modes
- 2015
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397. ; 27:1, s. 118-127
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Tidskriftsartikel (refereegranskat)abstract
- The majority of efforts on microbial and photosynthetic microbial fuel cells are both curiosity driven and made to possibly meet the future growing demand for sustainable energy. The most metabolically versatile purple bacteria Rhodobacter capsulatus is a potential candidate for this purpose. However, utilizing bacteria in such systems requires efficient electronic transfer communication between the microbial cells and the electrodes, which is one of the greatest challenges. Previous studies demonstrated that osmium redox polymers (ORPs) could be used for extracellular electron transfer between the cells and electrodes. Recently, heterotrophically grown R. capsulatus has been wired with ORP modified electrodes. Here in this communication, we report electron transfer from R. capsulatus grown under heterotrophic as well as under photoheterotrophic conditions to electrodes. The cells, immobilized on bare graphite and ORP modified graphite electrodes, were excited with visible light and subsequent photosynthetic electron transfer was recorded using cyclic voltammetric and chronoamperometric measurements. Photoheterotrophically grown R. capsulatus cells on bare graphite generate a significant photocurrent density of 3.46 mu A cm(-2), whereas on an ORP modified electrode the current density increases to 8.46 mu A cm(-2). Furthermore, when 1 mM p-benzoquinone is added to the electrolyte the photocurrent density reaches 12.25 mu A cm(-2). Our results could have significant implications in photosynthetic energy conversion and in development of photobioelectrochemical devices.
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| 8. |
- Hasan, Kamrul, et al.
(författare)
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Electrochemical communication between heterotrophically grown Rhodobacter capsulatus with electrodes mediated by an osmium redox polymer.
- 2013
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Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 93:Online 15 June 2012, s. 30-36
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Tidskriftsartikel (refereegranskat)abstract
- The metabolically versatile purple bacteria Rhodobacter capsulatus was investigated to check its possible applicability in biofuel cells and electrochemical microbial biosensors. The wild type strain ATCC 17015 and mutant strain 37b4 lacking the lipopolysaccharide capsule was compared for their ability to communicate with electrodes modified with an osmium redox polymer. In this work, aerobic heterotrophically grown R. capsulatus were used to screen for efficient cell-electrode communication for later implementation using photoheterotrophically grown bacteria. The bacterial cells embedded in the osmium polymer matrix demonstrated efficient electrical "wiring" with the electrodes and were able to generate a noticeable current with succinate as substrate. Interestingly, at 2mM succinate the wild type strain showed much better bioelectrocatalytic current generation (4.25μA/cm(2)) than the strain lacking capsule (1.55μA/cm(2)). The wild type strain also exhibited a stable current response for longer time, demonstrating that the bacterial lipopolysaccharide in fact enhances the stability of the polymer matrix layer of the modified electrode. Control experiments with R. capsulatus without any mediator did not show any current irrespective of the capsule presence. This demonstrates that development of photosensors and other light driven bioelectrochemical devices could be feasible using R. capsulatus and will be at focus for future studies.
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| 9. |
- Leiding, Thom, et al.
(författare)
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Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.
- 2009
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 388, s. 296-305
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Tidskriftsartikel (refereegranskat)abstract
- Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet (UV)-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.
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| 10. |
- Li, Ping, et al.
(författare)
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PcoB is a defense outer membrane protein that facilitates cellular uptake of copper
- 2022
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 31:7
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Tidskriftsartikel (refereegranskat)abstract
- Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.
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