| 1. |
- Adolfsson, Dan E., 1989-, et al.
(författare)
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Enhanement of amyloid fibril binding by ring expansion of thiazolino fused 2-pyridone peptidomimetics
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Thiazolino fused 2-pyridones undergo thiazoline ring opening by reaction with 2-nitrobenzyl bromide through thi- oether attack, and base promoted fragmentation of the resulting sulfonium ions. Subsequent deprotonation of the benzylic carbon and intramolecular 1,4-addition leads to ring closure, generating dihydrothiazine fused 2-pyridones by net ring expansion of the thiazoline ring. Application of the ring expansion procedure to the pyridine and pyrimidine fused thiazolino 2-pyridones provided compounds with enhanced fibril binding activity.
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| 2. |
- Adolfsson, Dan E., 1989-, et al.
(författare)
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Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-β fibrils
- 2020
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Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 85:21, s. 14174-14189
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- A BF3×OEt2 catalyzed intramolecular Povarov reaction was used to synthesize a library of 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with a range of O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid β fibrils in vitro. Analogs substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.
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| 3. |
- Alam, Athar, et al.
(författare)
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Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence
- 2020
-
Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 16:4, s. 1-27
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Tidskriftsartikel (refereegranskat)abstract
- Author summary Type VI secretion systems (T6SSs) are essential virulence determinants of many Gram-negative pathogens, including Francisella tularensis. This highly virulent bacterium encodes an atypical T6SS lacking ClpV, the ATPase crucial for prototypic T6SS sheath disassembly. It, however, possesses ClpB, a protein critical for heat shock survival via its interaction with DnaK. Since ClpB possesses ATPase activity, it has been hypothesized to provide a compensatory function for the absence of ClpV, a hypothesis supported by the recent findings from us and others. Here, we investigated how F. tularensis ClpB controls T6S. In silico modelling of the ClpB-DnaK complex identified key interactions that were experimentally verified. For example, mutating one of the DnaK-interacting residues rendered the bacterium exquisitely susceptible to heat shock, but had no effect on T6S and virulence. In contrast, removing the N-terminal of ClpB only had a slight effect on the heat shock response, but strongly compromised both T6S and virulence. Intriguingly, the Escherichia coli ClpB could fully complement the function of F. tularensis ClpB. The data demonstrate that the two critical roles of ClpB, mediating heat shock survival and effective T6S, are dissociated and that the N-terminal is crucial for T6S and virulence. Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the Delta clpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.
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| 4. |
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| 5. |
- Bharate, Jaideep B., et al.
(författare)
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K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones
- 2021
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Ingår i: Organic and biomolecular chemistry. - : The Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 19:44, s. 9758-9772
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Tidskriftsartikel (refereegranskat)abstract
- We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature amyloid-β and α-synuclein fibrils.
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| 6. |
- Cairns, Andrew G., et al.
(författare)
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Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy
- 2018
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 9:11, s. 2542-2547
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Tidskriftsartikel (refereegranskat)abstract
- Previous work in our laboratories has identified a series of peptidomimetic 2-pyridone molecules as modulators of alpha-synuclein (α-syn) fibrillization in vitro. As a first step toward developing molecules from this scaffold as positron emission tomography imaging agents, we were interested in evaluating their blood-brain barrier permeability in nonhuman primates (NHP) in vivo. For this purpose, 2-pyridone 12 was prepared and found to accelerate α-syn fibrillization in vitro. Acid 12, and its acetoxymethyl ester analogue 14, were then radiolabeled with 11C (t1/2 = 20.4 min) at high radiochemical purity (>99%) and high specific radioactivity (>37 GBq/μmol). Following intravenous injection of each compound in NHP, a 4-fold higher radioactivity in brain was observed for [11C]14 compared to [11C]12 (0.8 vs 0.2 SUV, respectively). [11C]14 was rapidly eliminated from plasma, with [11C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein deposits in brain.
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| 7. |
- Chorell, Erik, et al.
(författare)
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Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:10, s. e0140194-
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson's disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.
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| 8. |
- Clifton, Luke A., et al.
(författare)
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Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis
- 2023
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:22
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Tidskriftsartikel (refereegranskat)abstract
- Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of whichwere critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death.
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| 9. |
- Clifton, Luke A., et al.
(författare)
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Insight into Bcl-2 proteins' functioning at mitochondrial membrane level
- 2023
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 122:3S1, s. 232a-232a
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Tidskriftsartikel (refereegranskat)abstract
- Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.
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| 10. |
- Dingeldein, Artur Peter Günther, et al.
(författare)
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Bax to the future : A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies
- 2019
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 158, s. 20-26
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.
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| 11. |
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| 12. |
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| 13. |
- Dulko-Smith, Beata, et al.
(författare)
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Mechanistic basis for a connection between the catalytic step and slow opening dynamics of adenylate kinase
- 2023
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 63:5, s. 1556-1569
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.
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| 14. |
- Esteban-Martin, Santiago, et al.
(författare)
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Correlated Inter-Domain Motions in Adenylate Kinase
- 2014
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Ingår i: PloS Computational Biology. - : PLOS. - 1553-734X .- 1553-7358. ; 10:7, s. e1003721-
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Tidskriftsartikel (refereegranskat)abstract
- Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.
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| 15. |
- Evans, Margery L, et al.
(författare)
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The bacterial curli system possesses a potent and selective inhibitor of amyloid formation
- 2015
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-4164 .- 1097-2765. ; 57:3, s. 445-455
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Tidskriftsartikel (refereegranskat)abstract
- Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.
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| 16. |
- Jain, Neha, et al.
(författare)
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Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:46, s. 12184-12189
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Tidskriftsartikel (refereegranskat)abstract
- During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer’s-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.
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| 17. |
- Kelly, Rachel, et al.
(författare)
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The Small Molecule Alpha-Synuclein Aggregator, FN075, Enhances Alpha-Synuclein Pathology in Subclinical AAV Rat Models
- 2021
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Animal models of Parkinson’s disease, in which the human α-synuclein transgene is overexpressed in the nigrostriatal pathway using viral vectors, are widely considered to be the most relevant models of the human condition. However, although highly valid, these models have major limitations related to reliability and variability, with many animals exhibiting pronounced α-synuclein expression failing to demonstrate nigrostriatal neurodegeneration or motor dysfunction. Therefore, the aim of this study was to determine if sequential intra-nigral administration of AAV-α-synuclein followed by the small α-synuclein aggregating molecule, FN075, would enhance or precipitate the associated α-synucleinopathy, nigrostriatal pathology and motor dysfunction in subclinical models. Rats were given unilateral intra-nigral injections of AAV-α-synuclein (either wild-type or A53T mutant) followed four weeks later by a unilateral intra-nigral injection of FN075, after which they underwent behavioral testing for lateralized motor functionality until they were sacrificed for immunohistological assessment at 20 weeks after AAV administration. In line with expectations, both of the AAV vectors induced widespread overexpression of human α-synuclein in the substantia nigra and striatum. Sequential administration of FN075 significantly enhanced the α-synuclein pathology with increased density and accumulation of the pathological form of the protein phosphorylated at serine 129 (pS129-α-synuclein). However, despite this enhanced α-synuclein pathology, FN075 did not precipitate nigrostriatal degeneration or motor dysfunction in these subclinical AAV models. In conclusion, FN075 holds significant promise as an approach to enhancing the α-synuclein pathology in viral overexpression models, but further studies are required to determine if alternative administration regimes for this molecule could improve the reliability and variability in these models.
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| 18. |
- Kovermann, Michael, et al.
(författare)
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Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- 2015
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Ingår i: Nature Communications. - : Macmillan Publishers Ltd.. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
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| 19. |
- Mikaelsson, Therese, et al.
(författare)
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Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding
- 2013
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 104:3, s. 694-704
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Tidskriftsartikel (refereegranskat)abstract
- Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.
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| 20. |
- Mikaelsson, Therese, et al.
(författare)
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Effects of macromolecular crowding on folded protein homologs: global versus local consequences
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Proteins function in cellular environments that are crowded with other biomolecules. This reduction of available space may change biophysical properties of proteins as compared to dilute solutions in vitro. Here we have investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. First, as expected for an excluded volume effect, the thermal stability increased for S16Meso in the presence of dextran 20. In accord with a dominating entropic effect, chemical denaturation experiments at different fixed temperatures showed that the crowding effect was temperature independent. Far-UV circular dichroism spectra did not reveal any secondary structural changes in the folded proteins in presence of dextran 20. However, Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY in S16Meso depend on the level of crowding agent. The BODIPY-group was attached at three specific positions in S16Meso. All S16Meso variants exhibited a decrease in the average distance up to 100 mg/mL dextran 20, indicating folded-state compaction, whereas the change in distance became anisotropic at higher dextran concentrations. In contrast, the two S16Thermo mutants, that are thermodynamically more stable than the mesophilic variants, did not show any change in distance upon increasing dextran 20 concentrations. Notably, the BODIPY fluorescence quantum yields and lifetimes of the two homologs decreased gradually in the presence of dextran 20. To investigate the origin of this, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labelled dextran. The results reveal quenching between tyrosine and BODIPY at all concentrations of Tyr-dextran. The data also suggests that dynamic local interactions between protein and crowding agent can occur at some conditions.
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| 21. |
- Mikaelsson, Therese, et al.
(författare)
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Macromolecular crowding effects on two homologs of ribosomal protein S16 : protein-dependent structural changes and local interactions
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 107:2, s. 401-410
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Tidskriftsartikel (refereegranskat)abstract
- Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16T(herme)) and mesophilic (S161homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic Meso, protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Forster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16 Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16me, allowing measurements of three intraprotein distances. All S16meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two 516-rhermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.
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| 22. |
- Mondol, Tanumoy, et al.
(författare)
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Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B
- 2016
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 470:3, s. 663-669
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Tidskriftsartikel (refereegranskat)abstract
- Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.
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| 23. |
- Nadeem, Aftab, et al.
(författare)
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Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae
- 2022
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.
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| 24. |
- Nilsson, Lina, et al.
(författare)
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Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein
- 2013
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 117:42, s. 13038-13050
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Tidskriftsartikel (refereegranskat)abstract
- Although strictly regulated, pH and solute concentrations in cells may exhibit temporal and spatial fluctuations. Here we study the effect of such changes on the stability, structure, and dynamics in vitro and in silico of a two-domain construct (WD56) of the fifth and sixth metal-binding domains of the copper transport protein, ATP7B (Wilson disease protein). We find that the thermal stability of WD56 is increased by 40 °C when increasing the pH from 5.0 to 7.5. In contrast, addition of salt at pH 7.2 decreases WD56 stability by up to 30 °C. In agreement with domain-domain coupling, fractional copper loading increases the stability of both domains. HSQC chemical shift changes demonstrate that, upon lowering the pH from 7.2 to 6, both His in WD6 as well as the second Cys of the copper site in each domain become protonated. MD simulations reveal increased domain-domain fluctuations at pH 6 and in the presence of high salt concentration, as compared to at pH 7 and low salt concentration. Thus, the surface charge distribution at high pH contributes favorably to overall WD56 stability. By introducing more positive charges by lowering the pH, or by diminishing charge-charge interactions by salt, fluctuations among the domains are increased and thereby overall stability is reduced. Copper transfer activity also depends on pH: delivery of copper from chaperone Atox1 to WD56 is more efficient at pH 7.2 than at pH 6 by a factor of 30. It appears that WD56 is an example where the free energy landscapes for folding and function are linked via structural stability.
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| 25. |
- Näsström, Thomas, et al.
(författare)
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A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations
- 2020
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Ingår i: International Journal of Molecular Sciences. - Basel, Switzerland : MDPI. - 1661-6596 .- 1422-0067. ; 21:5
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Tidskriftsartikel (refereegranskat)abstract
- Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.
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