| 1. |
- Everberg, Henrik, et al.
(author)
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Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis
- 2006
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1122:1-2, s. 35-46
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Journal article (peer-reviewed)abstract
- It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies. (c) 2006 Elsevier B.V. All rights reserved.
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| 2. |
- Frankel, Rebecca, et al.
(author)
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Autocatalytic amplification of Alzheimer-associated Aβ42 peptide aggregation in human cerebrospinal fluid
- 2019
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 2:1
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Journal article (peer-reviewed)abstract
- Alzheimer’s disease is linked to amyloid β (Aβ) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aβ aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aβ42 in human CSF through kinetic experiments at several Aβ42 monomer concentrations (0.8–10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.
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| 3. |
- Gustavsson, Tobias, et al.
(author)
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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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In: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 19:8, s. 1445-1460
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Journal article (peer-reviewed)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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| 4. |
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| 5. |
- Leiding, Thom, et al.
(author)
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Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.
- 2009
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In: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 388, s. 296-305
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Journal article (peer-reviewed)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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| 6. |
- Leiding, Thom
(author)
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Probing Proton Translocation in Influenza A/M2 Proteoliposomes - A systematic Approach to Membrane-Protein Reconstitutions
- 2010
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Doctoral thesis (other academic/artistic)abstract
- An improved method for reconstituting membrane proteins into artificial liposomes for quantitative functional analysis is presented. A number of key parameters for reconstitution by detergent removal are assessed in this thesis: The lipid-to-protein ratio, the detergent-to-lipid ratio and the lipid and cholesterol composition. New porphyrin-based pH-probes are evaluated. Based on this systematic, comprehensive approach to protein reconstitution, we present a robust system for quantitative proton-flux analysis, as demonstrated by influenza virus A M2 reconstitution into large unilamellar vesicles. The M2 protein is a small, single-span transmembrane protein, which plays an important role in the life cycle of influenza A virus and is the target of the adamantane series of anti-influenza drugs. This virus enters cells via the endosomes; as the endosomes acidify M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation, which is required to effect a significant change in the internal effective pH. Here, we show that M2 has essential antiporter-like activity. This should lead to future investigations of the biophysical mechanism of transport, which will have implications for the design of new generations of M2-targeting drugs as well as furthering our understanding of cotransport.
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| 7. |
- Leiding, Thom, et al.
(author)
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Proton and cation transport activity of the M2 proton channel from influenza A virus.
- 2010
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 107:35, s. 15409-15414
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Journal article (peer-reviewed)abstract
- The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.
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| 8. |
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| 9. |
- Ortigosa-Pascual, Lei, et al.
(author)
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Photo-Induced Cross-Linking of Unmodified α-Synuclein Oligomers
- 2023
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In: ACS Chemical Neuroscience. - 1948-7193. ; 14:17, s. 3192-3205
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Journal article (peer-reviewed)abstract
- Photo-induced cross-linking of unmodified proteins (PICUP) has been used in the past to study size distributions of protein assemblies. PICUP may, for example, overcome the significant experimental challenges related to the transient nature, heterogeneity, and low concentration of amyloid protein oligomers relative to monomeric and fibrillar species. In the current study, a reaction chamber was designed, produced, and used for PICUP reaction optimization in terms of reaction conditions and lighting time from ms to s. These efforts make the method more reproducible and accessible and enable the use of shorter reaction times compared to previous studies. We applied the optimized method to an α-synuclein aggregation time course to monitor the relative concentration and size distribution of oligomers over time. The data are compared to the time evolution of the fibril mass concentration, as monitored by thioflavin T fluorescence. At all time points, the smaller the oligomer, the higher its concentration observed after PICUP. Moreover, the total oligomer concentration is highest at short aggregation times, and the decline over time follows the disappearance of monomers. We can therefore conclude that these oligomers form from monomers.
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| 10. |
- Pálmadóttir, Tinna, et al.
(author)
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Charge Regulation during Amyloid Formation of α-Synuclein
- 2021
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:20, s. 7777-7791
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Journal article (peer-reviewed)abstract
- Electrostatic interactions play crucial roles in protein function. Measuring pKa value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pKa value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pKaave value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pKaave values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein.
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