| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- Haglund, Ellinor, et al.
(författare)
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The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway
- 2009
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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. ; 106:51, s. 21619-21624
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Tidskriftsartikel (refereegranskat)abstract
- An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.
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| 5. |
- Hedberg, Linda, et al.
(författare)
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Scattered Hammond plots reveal second level of site-specific information in protein folding: ′ (β‡)
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America PNAS. - : Proceedings of the National Academy of Sciences. ; 101:20, s. 7606–11-
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Tidskriftsartikel (refereegranskat)abstract
- Site-specific information about structural heterogeneities of the protein-folding transition-state ensemble is commonly derived from the scatter of the Brønsted plot through the individual values of = Δlogkf/ΔlogKD-N. Here, we provide a second level of site-specific detail in the transition-state analysis by demonstrating that the scatter of the Hammond plot is related to heterogeneities in the -value growth. That is, the extent of transition-state movement (Δβ‡) is proportional to the free-energy gradient of the mutational perturbation across the top of the activation barrier, ′(β‡) ΔlogKD-N. The analysis is applied to the two-state protein L23 where the site-specific free-energy gradients are used to identify the interactions that show the highest degree of consolidation after crossing the barrier top. These interactions are distributed as a shell around the high- initiation point and denote the side-chain contacts that add criticality to the folding nucleus.
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| 6. |
- Hubner, Isaac A, et al.
(författare)
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Simulation, experiment, and evolution: Understanding nucleation in protein S6 folding
- 2004
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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. ; 101:22, s. 8354-9
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we explore nucleation and the transition state ensemble of the ribosomal protein S6 using a Monte Carlo (MC) Go model in conjunction with restraints from experiment. The results are analyzed in the context of extensive experimental and evolutionary data. The roles of individual residues in the folding nucleus are identified, and the order of events in the S6 folding mechanism is explored in detail. Interpretation of our results agrees with, and extends the utility of, experiments that shift -values by modulating denaturant concentration and presents strong evidence for the realism of the mechanistic details in our MC Go model and the structural interpretation of experimental -values. We also observe plasticity in the contacts of the hydrophobic core that support the specific nucleus. For S6, which binds to RNA and protein after folding, this plasticity may result from the conformational flexibility required to achieve biological function. These results present a theoretical and conceptual picture that is relevant in understanding the mechanism of nucleation in protein folding.
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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
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