| 1. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro
- 2013
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 105:7, s. 1689-1699
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Tidskriftsartikel (refereegranskat)abstract
- Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.
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| 2. |
- Homouz, Dirar M., et al.
(författare)
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Role of Zero-Order Loop in Protein Unfolding Case Study with Apoazurin
- 2020
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:3, Supplement 1, s. 197A-197A
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Konferensbidrag (refereegranskat)abstract
- Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) is a two-state folder that has a single disulfide bond between residues 3 and 26. This bond covalently connects the N-termini of beta-strands 1 and 3; thereby it creates a zero-order loop. The loop restricts the conformational space for the apoazurin polypeptide. In order to understand the role played by the zero-order loop, we used molecular dynamics (MD) simulations to compare two variants of apoazurin; one variant called “loop” which contained the disulfide and another called “open” in which the disulfide bond between residues 3 and 26 was removed. MD simulations were performed to probe the unfolding pathway and stability of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the folded structure apoazurin is somewhat more stable due to the presence of the disulfide bond. However, the disulfide bond plays a prominent role in the apoazurin unfolding mechanism: we find that it changes both the folding-transition state and the unfolded-state ensemble of conformations.
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| 3. |
- Horvath, Istvan, 1979, et al.
(författare)
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Distinct growth regimes of α-synuclein amyloid elongation
- 2023
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 122:12, s. 2556-2563
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Tidskriftsartikel (refereegranskat)abstract
- Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in β-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.
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| 4. |
- Horvath, Istvan, 1979, et al.
(författare)
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Macromolecular crowding modulates α-synuclein amyloid fiber growth
- 2021
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 120:16, s. 3374-3381
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Tidskriftsartikel (refereegranskat)abstract
- The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson's disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.
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| 5. |
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| 6. |
- Kahra, Dana, et al.
(författare)
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The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1
- 2016
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 110:1, s. 95-102
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Tidskriftsartikel (refereegranskat)abstract
- Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10-14 M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.
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| 7. |
- Köhn, Birgit, et al.
(författare)
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A Luminal Loop of Wilson Disease Protein Binds Copper and Is Required for Protein Activity
- 2018
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 115:6, s. 1007-1018
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Tidskriftsartikel (refereegranskat)abstract
- The copper-transporting ATPase ATP7B is essential for loading of copper ions to copper-dependent enzymes in the secretory pathway; its inactivation results in Wilson disease. In contrast to copper-ion uptake by the cytoplasmic domains, ATP7B-mediated copper-ion release in the Golgi has not been explored yet. We demonstrate here that a luminal loop in ATP7B, rich in histidine/methionine residues, binds reduced copper (Cu(I)) ions, and identified copper-binding residues play an essential role in ATP7B-mediated metal ion release. NMR experiments on short-peptide models demonstrate that three methionine and two histidine residues are specifically involved in Cu(I) ion binding; with these residues replaced by alanines, no Cu(I) ion interaction is detected. Although more than one Cu(I) ion can interact with the wild-type peptide, removing either all histidine or all methionine residues reduces the stoichiometry to one Cu(I) ion binding per peptide. Using a yeast complementation assay, we show that for efficient copper transport by full-length ATP7B, the complete set of histidine and methionine residues in the lumen loop are required. The replacement of histidine/methionine residues by alanines does not perturb overall ATP7B structure, as the localization of ATP7B variants in yeast cells matches that of the wild-type protein. Thus, in similarity to ATP7A, ATP7B also appears to have a luminal “exit” copper ion site.
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| 8. |
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| 9. |
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| 10. |
- Wittung Stafshede, Pernilla, 1968
(författare)
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Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm
- 2016
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 110:3, s. 179A-179A
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Tidskriftsartikel (refereegranskat)abstract
- Although copper (Cu) is an essential metal for most living organisms, highlevels and free such ions are toxic. In humans, after cellular uptake via themembrane-bound importer Ctr1, Cu is transported to targets by cytoplasmicCu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases(i.e., ATP7B or Wilson disease protein) in the Golgi (secretory path; here,most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cuspecifically to cytoplasmic superoxide dismutase. In contrast to bacterial andyeast homologs, ATP7B has six similar metal-binding domains protrudinginto the cytoplasm: possibly, conformational changes among these regulateoverall ATP7B activity. To reveal underlying molecular mechanisms as wellas thermodynamic and kinetic driving forces for human Cu transport - fromthe cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified proteins, domain constructs and engineered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceedsthrough Cu-bridged hetero-protein dimers displaying enthalpy-entropycompensation, (d) conformational changes and domain-domain interactionswithin ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding proteins in a yeast two-hybrid screen.
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