| 1. |
- Abelein, Axel, et al.
(författare)
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Zinc as chaperone-mimicking agent for retardation of amyloid beta peptide fibril formation
- 2015
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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. ; 112:17, s. 5407-5412
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Tidskriftsartikel (refereegranskat)abstract
- Metal ions have emerged to play a key role in the aggregation process of amyloid beta (A beta) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic A beta-Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn2+ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc A beta(40) aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn2+, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn2+ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded A beta(40) species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded A beta(40)-Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for A beta(40).
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| 2. |
- Bergh, Johan, 1983-, et al.
(författare)
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Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:14, s. 4489-4494
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Tidskriftsartikel (refereegranskat)abstract
- Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.
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| 3. |
- Danielsson, Jens, et al.
(författare)
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Global structural motions from the strain of a single hydrogen bond
- 2013
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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. ; 110:10, s. 3829-3834
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Tidskriftsartikel (refereegranskat)abstract
- The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the beta-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?
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| 4. |
- Danielsson, Jens, et al.
(författare)
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Thermodynamics of protein destabilization in live cells
- 2015
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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. ; 112:40, s. 12402-12407
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Tidskriftsartikel (refereegranskat)abstract
- Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized interaction landscape of the cellular interior.
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| 5. |
- Kurnik, Martin, et al.
(författare)
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Folding without charges
- 2012
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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. ; 109:15, s. 5705-5710
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Tidskriftsartikel (refereegranskat)abstract
- Surface charges of proteins have in several cases been found to function as structural gatekeepers, which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.
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| 6. |
- Lang, Lisa, et al.
(författare)
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Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium
- 2012
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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. ; 109:44, s. 17868-17873
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Tidskriftsartikel (refereegranskat)abstract
- Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.
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| 7. |
- Lang, Lisa, et al.
(författare)
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SOD1 aggregation in ALS mice shows simplistic test tube behavior
- 2015
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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. ; 112:32, s. 9878-9883
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Tidskriftsartikel (refereegranskat)abstract
- A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.
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| 8. |
- Mu, Xin, et al.
(författare)
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Physicochemical code for quinary protein interactions in Escherichia coli
- 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. ; 114:23, s. E4556-E4563
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Tidskriftsartikel (refereegranskat)abstract
- How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical-chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.
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| 9. |
- Nordlund, Anna, et al.
(författare)
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Functional features cause misfolding of the ALS-provoking enzyme SOD1
- 2009
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 106:24, s. 9667-9672
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Tidskriftsartikel (refereegranskat)abstract
- The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn2+ loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a non-native Zn2+ ion with mu M affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn2+ ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features.
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| 10. |
- Wang, Huabing, et al.
(författare)
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Exposing the distinctive modular behavior of β-strands and α-helices in folded proteins
- 2020
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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. ; 117:46, s. 28775-28783
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Tidskriftsartikel (refereegranskat)abstract
- Although folded proteins are commonly depicted as simplistic combinations of β-strands and α-helices, the actual properties and functions of these secondary-structure elements in their native contexts are just partly understood. The principal reason is that the behavior of individual β- and α-elements is obscured by the global folding cooperativity. In this study, we have circumvented this problem by designing frustrated variants of the mixed α/β-protein S6, which allow the structural behavior of individual β-strands and α-helices to be targeted selectively by stopped-flow kinetics, X-ray crystallography, and solution-state NMR. Essentially, our approach is based on provoking intramolecular "domain swap." The results show that the α- and β-elements have quite different characteristics: The swaps of β-strands proceed via global unfolding, whereas the α-helices are free to swap locally in the native basin. Moreover, the α-helices tend to hybridize and to promote protein association by gliding over to neighboring molecules. This difference in structural behavior follows directly from hydrogen-bonding restrictions and suggests that the protein secondary structure defines not only tertiary geometry, but also maintains control in function and structural evolution. Finally, our alternative approach to protein folding and native-state dynamics presents a generally applicable strategy for in silico design of protein models that are computationally testable in the microsecond–millisecond regime.
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