| 1. |
- Khan, Ashhar, et al.
(author)
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Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant
- 2017
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 8:9, s. 2019-2026
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Journal article (peer-reviewed)abstract
- Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.
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| 2. |
- Rutsdottir, Gudrun, et al.
(author)
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Structural model of dodecameric heat-shock protein Hsp21 : Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity
- 2017
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 292:19, s. 8103-8121
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Journal article (peer-reviewed)abstract
- Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
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| 3. |
- Weininger, Ulrich, et al.
(author)
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Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected (13)C CPMG relaxation dispersion.
- 2012
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In: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 54:1, s. 9-14
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Journal article (peer-reviewed)abstract
- Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.
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| 4. |
- Weininger, Ulrich, et al.
(author)
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Slow Aromatic Ring Flips Detected Despite Near-Degenerate NMR Frequencies of the Exchanging Nuclei.
- 2013
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In: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 117:31, s. 9241-9247
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Journal article (peer-reviewed)abstract
- Aromatic ring flips of Phe and Tyr residues are a hallmark of protein dynamics with a long history in molecular biophysics. Ring flips lead to symmetric exchange of nuclei between sites with distinct magnetic environments, which can be probed by NMR spectroscopy. Current knowledge of ring-flip rates originates from rare cases in which the chemical shift difference between the two sites is sufficiently large and the ring-flip rate sufficiently slow, typically kflip < 10(3) s(-1), so that separate peaks are observed in the NMR spectrum for the two nuclei, enabling direct measurement of the flip rate. By contrast, a great majority of aromatic rings show single peaks for each of the pairs of δ or ε nuclei, which commonly are taken as inferential evidence that the flip rate is fast, kflip ≫ 10(3) s(-1), even though rate measurements have not been achieved. Here we report a novel approach that makes it possible to identify slow ring flips in previously inaccessible cases where only single peaks are observed. We demonstrate that Y21 in the bovine pancreatic trypsin inhibitor (BPTI) has a slow ring-flip rate, kflip < 100 s(-1), a result that contrasts with previous estimates of 10(4)-10(6) s(-1) inferred from the single-peak spectrum of Y21. Comparison with a recent 1 ms molecular dynamics trajectory of BPTI shows qualitative agreement and highlights the value of accurate aromatic ring flip data as an important benchmark for molecular dynamics simulations of proteins across wide time scales.
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