| 1. |
- Cabaleiro-Lago, Celia, et al.
(författare)
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Inhibition of IAPP and IAPP(20-29) fibrillation by polymeric nanoparticles
- 2010
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Ingår i: Langmuir. - : The American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 26:5, s. 3453-3461
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Tidskriftsartikel (refereegranskat)abstract
- The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.
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| 2. |
- Hellstrand, Erik, et al.
(författare)
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Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.
- 2013
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 280:11, s. 2675-2687
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Tidskriftsartikel (refereegranskat)abstract
- This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.
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| 3. |
- Linse, Björn, et al.
(författare)
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Monte Carlo simulations of protein amyloid formation reveal origin of sigmoidal aggregation kinetics.
- 2011
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Ingår i: Molecular BioSystems. - : Royal Society of Chemistry (RSC). - 1742-2051 .- 1742-206X. ; 7, s. 2296-2303
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Tidskriftsartikel (refereegranskat)abstract
- Severe conditions and lack of cure for many amyloid diseases make it highly desired to understand the underlying principles of formation of fibrillar aggregates (amyloid). Here, amyloid formation from peptides was studied using Monte Carlo simulations. Systems of 20, 50, 100, 200 or 500 hexapeptides were simulated. Association kinetics were modeled equal for fibrillar and other (inter- and intra-peptide) contacts and assumed to be faster the lower the effective contact order, which represents the distance in space. Attempts to form contacts were thus accepted with higher probability the lower the effective contact order, whereby formation of new contacts next to preexisting ones is favored by shorter physical separation. Kinetic discrimination was invoked by using two different life-times for formed contacts. Contacts within amyloid fibrils were assumed to have on average longer life-time than other contacts. We find that the model produces fibrillation kinetics with a distinct lag phase, and that the fibrillar contacts need to dissociate on average 5-20 times slower than all other contacts for the fibrillar structure to dominate at equilibrium. Analysis of the species distribution along the aggregation process shows that no other intermediate is ever more populated than the dimer. Instead of a single nucleation event there is a concomitant increase in average aggregate size over the whole system, and the occurrence of multiple parallel processes makes the process more reproducible the larger the simulated system. The sigmoidal shape of the aggregation curves arises from cooperativity among multiple interactions within each pair of peptides in a fibril. A governing factor is the increasing probability as the aggregation process proceeds of neighboring reinforcing contacts. The results explain the very strong bias towards cross β-sheet fibrils in which the possibilities for cooperativity among interactions involving neighboring residues and the repetitive use of optimal side-chain interactions are explored at maximum.
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| 4. |
- Linse, Sara, et al.
(författare)
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Protein folding through kinetic discrimination
- 2007
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:27, s. 8481-8486
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Tidskriftsartikel (refereegranskat)abstract
- Proteins fold on a mu s-ms time scale. However, the number of possible conformations of the polypeptide backbone is so large that random sampling would not allow the protein to fold within the lifetime of the universe, the Levinthal paradox. We show here that a protein chain can fold efficiently with high fidelity if on average native contacts survive longer than non-native ones, that is, if the dissociation rate constant for breakage of a contact is lower for native than for non-native interactions. An important consequence of this finding is that no pathway needs to be specified for a protein to fold. Instead, kinetic discrimination among formed contacts is a sufficient criterion for folding to proceed to the native state. Successful protein folding requires that productive contacts survive long enough to obtain a certain level of probability that other native contacts form before the first interacting unit dissociates. If native contacts survive longer than non-native ones, this prevents misfolding and provides the folding process with directionality toward the native state. If on average all contacts survive equally long, the protein chain is deemed to fold through random search through all possible conformations (i.e., the Levinthal paradox). A modest degree of cooperativity among the native contacts, that is, decreased dissociation rate next to neighboring contacts, shifts the required ratio of dissociation rates into a realistic regime and makes folding a stochastic process with a nucleation step. No kinetic discrimination needs to be invoked in regards to the association process, which is modeled as dependent on the diffusion rate of chain segments.
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| 5. |
- O'Connell, David, et al.
(författare)
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Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 9:6, s. 1118-1132
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) = 1 muM) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments, of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff = 10(3) s(-1)) and high affinity (K(D) = 1 muM), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We have developed a microarray of the identified target proteins with which we can characterise the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage gated channel Kv6.1, (residues 474-493), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415).
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| 6. |
- Szczepankiewicz, Olga, et al.
(författare)
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N-Terminal Extensions Retard Aβ42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aβ42.
- 2015
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 137:46, s. 14673-14685
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid β-protein (Aβ) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid β-protein (Aβ) variants were believed to begin at or after the canonical β-secretase cleavage site within the amyloid β-protein precursor. However, N-terminally extended forms of Aβ (NTE-Aβ) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aβ42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aβ42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aβ42s form fibrils via the same mechanism as Aβ42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aβ42 and NTE-Aβ42 coaggregate to form mixed fibrils and fibrils of either Aβ42 or NTE-Aβ42 catalyze aggregation of all monomers. NTE-Aβ42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.
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| 7. |
- Abou-Hachem, Maher, et al.
(författare)
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Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
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Tidskriftsartikel (refereegranskat)abstract
- Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
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| 8. |
- Abou-Hachem, Maher, et al.
(författare)
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The modular organisation and stability of a thermostable family 10 xylanase
- 2003
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 21:5-6, s. 253-260
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.
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| 9. |
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| 10. |
- Andersson, Alexandra, et al.
(författare)
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Single-vesicle intensity and colocalization fluorescence microscopy to study lipid vesicle fusion, fission, and lipid exchange
- 2022
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Ingår i: Frontiers in Molecular Neuroscience. - : Frontiers Media SA. - 1662-5099. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties.
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