| 1. |
- Sahin, Cagla, et al.
(author)
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Structural basis for dityrosine-mediated inhibition of alpha-synuclein fibrillation
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Other publication (other academic/artistic)abstract
- α-synuclein (aSyn) is a small intrinsically disordered protein which can self-assemble into highly organized β-sheet structures that are found to accumulate in plaques in the brain of Parkinson’s Disease patients. Oxidative stress has been shown to be important for aSyn and its self-assembly. Here we characterize the molecular and structural effects that mild oxidation has on aSyn monomer and its aggregation. Using a combination of biophysical methods, SAXS and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages that reduce aSyn’s size by a factor of √2. MD simulations support our experimental results showing a stable and compact aSyn conformation that prevents self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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| 2. |
- Sahin, Cagla, et al.
(author)
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Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization
- 2022
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:27, s. 11949-11954
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Journal article (peer-reviewed)abstract
- α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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| 3. |
- Abramsson, Mia L, et al.
(author)
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Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry
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Other publication (other academic/artistic)abstract
- The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.
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| 4. |
- Abramsson, Mia L., et al.
(author)
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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
- 2021
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In: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:12, s. 2385-2393
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Journal article (peer-reviewed)abstract
- In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.
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| 5. |
- Allison, Timothy M., et al.
(author)
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Complementing machine learning‐based structure predictions with native mass spectrometry
- 2022
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In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6
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Journal article (peer-reviewed)abstract
- The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
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| 6. |
- Andersson, Marlene, et al.
(author)
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Biomimetic spinning of artificial spider silk from a chimeric minispidroin
- 2017
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In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 13:3, s. 262-
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Journal article (peer-reviewed)abstract
- Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
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| 7. |
- Andersson, Marlene, et al.
(author)
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Biomimetic spinning of artificial spider silk from a chimeric minispidroin
- 2017
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 254
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Journal article (peer-reviewed)abstract
- Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
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| 8. |
- Andersson, Marlene, et al.
(author)
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Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains
- 2014
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In: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 12:8, s. e1001921-
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Journal article (peer-reviewed)abstract
- Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.
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| 9. |
- Arndt, Tina, et al.
(author)
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Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Journal article (peer-reviewed)abstract
- Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.
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| 10. |
- Bolla, Jani Reddy, et al.
(author)
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A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins
- 2020
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In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 59:9, s. 3523-3528
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Journal article (peer-reviewed)abstract
- Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased k(off) rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.
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